Compstatin Analogs With Increased Solubility And Improved Pharmacokinetic Properties

ABSTRACT

Compounds comprising peptides capable of binding C3 protein and inhibiting complement activation are disclosed. The compounds comprise compstatin analogs in which the N-terminus and/or C-terminus contains an added component that improves (1) the peptide&#39;s solubility at physiological pH; (2) the peptide&#39;s plasma half-life; (3) the peptide&#39;s intraocular retention; and/or (4) the peptide&#39;s binding affinity to C3 or its fragments as compared to an unmodified compstatin peptide under equivalent conditions. Pharmaceutical compositions and methods of using the compounds are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

Continuation of PCT/US2019/026040, filed Apr. 5, 2019, which claimsbenefit of the filing date of U.S. Provisional Application No.62/654,055, filed Apr. 6, 2018, the entire contents of each of which areincorporated by reference herein.

GOVERNMENT SUPPORT

This invention was made with government support under grant numbersAI030040 and AI068730 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to activation of the complement cascade in thebody. In particular, this invention provides compstatin analogs thatbind the C3 protein with nanomolar affinity and exhibit robustcomplement inhibitory activity, increased solubility at physiologicalpH, plasma stability and in vivo retention.

BACKGROUND OF THE INVENTION

Various publications, including patents, published applications,technical articles and scholarly articles are cited throughout thespecification. Each of these cited publications is incorporated byreference herein, in its entirety.

The human complement system is a powerful player in the defense againstpathogenic organisms and the mediation of immune responses. Complementcan be activated through three different pathways: the classical,lectin, and alternative pathways. The major activation event that isshared by all three pathways is the proteolytic cleavage of the centralprotein of the complement system, C3, into its activation products C3aand C3b by C3 convertases. Generation of these fragments leads to theopsonization of pathogenic cells by C3b and iC3b, a process that rendersthem susceptible to phagocytosis or clearance, and to the activation ofimmune cells through an interaction with complement receptors.Deposition of C3b on target cells also induces the formation of newconvertase complexes and thereby initiates a self-amplification loop.

An ensemble of plasma and cell surface-bound proteins carefullyregulates complement activation to prevent host cells from self-attackby the complement cascade. However, excessive activation orinappropriate regulation of complement can lead to a number ofpathologic conditions, ranging from autoimmune to inflammatory diseases.The development of therapeutic complement inhibitors is therefore highlydesirable. In this context, C3 and C3b have emerged as promising targetsbecause their central role in the cascade allows for the simultaneousinhibition of the initiation, amplification, and downstream activationof complement.

As the number of clinical conditions linked to aberrant activation ofthe complement system continues to grow, so must the field of complementtherapeutics in order to meet the increasing demand for effective anddisease-tailored treatments. Despite tremendous ongoing efforts inresearch and drug development in this field, the firstcomplement-targeting drug, eculizumab (Soliris®, Alexion), remains theonly therapeutic on the market more than a decade after its FDA approvalfor the treatment of paroxysmal nocturnal hemoglobinuria (PNH).Eculizumab is a humanized monoclonal antibody against C5 that preventsits cleavage into C5a and C5b, thus blocking activation of the terminalpathway of complement. Thus, there remains a need to identify additionalcomplement-targeting compounds as potential therapeutics.

Upstream intervention at the level of C3, the central component of thecomplement cascade, has been explored as an intervention approach, giventhe involvement of C3 in a variety of pathogenic pathways. Thecompstatin family, a group of cyclic peptides consisting of about 13amino acids, was initially introduced two decades ago and shows strongbinding affinity toward C3 from humans and non-human primates (NHPs)(Sahu et al., 1996, The Journal of Immunology 157:884-891). Continuousresearch and further development of compstatin has resulted in a numberof next-generation analogs with improved complement inhibitory activityand target affinity, including the most potent derivative, Cp40 (see Quet al., 2011, Mol Immunol 48:481-489; Qu et al., 2013, Immunobiology218:496-505). As described by Qu et al. (2013, supra), Cp40 has asubnanomolar binding affinity for C3, almost 6,000-fold higher than thatof the parent peptide, and an extended plasma half-life. However,despite its high solubility in water, Cp40 shows less solubility atphysiological pH, thus limiting the routes of administration that can beused to deliver effective amounts of Cp40 as well as potentially causingincreased precipitation at the injection site resulting in localirritation or pain to the patient. Further, a compstatin analog withhigh solubility at physiological pH would improve its suitability forboth intravenous and subcutaneous administration, the latter providingvarious benefits over intravenous injection, such as lowering the costfor health care systems, reducing the frequency of administration,increasing the convenience and compliance for patients, and providingmore options for self-administration.

Peptide modifications have been used to enhance solubility and extendthe half-life of compounds in vivo. However, such modifications candecrease compound activity and/or binding, which would reduce thecompound's beneficial characteristics. PEGylation has been used topotentiate drugs with undesirable properties, and these PEGylatedcompounds tend to display enhanced solubility. However, the improvedsolubility conferred by PEGylation may come at a cost as preserving thepharmacological activity of these modified compounds remains a challengein some cases, thereby limiting the therapeutic benefit of certaincompounds (for example, see Zhang et al., 2014, Expert Opin Drug MegabToxicol 10:1691-1702). Indeed while PEGylation has been successfullyapplied to extend the plasma half-life and solubility of Cp40, it hasbeen reported that a Cp40 coupled to an mPEG(40k) exhibited more than a100-fold decrease in binding affinity for C3 fragments and a drop ininhibitory activity (Risitano et al., 2014, Blood 123:2019-2101).Further, with respect to in vivo drug clearance, it has been reportedthat clearance of PEG from the body decreases proportionately to thesize of the PEG (see, e.g., Webster et al., 2007, Drug Megab & Dispos35: 9-16).

In view of the foregoing, it is clear that the development of modifiedcompstatin peptides with greater activity, in vivo stability, plasmaresidence time, solubility and/or bioavailability would constitute asignificant advance in the art.

SUMMARY OF THE INVENTION

The present invention provides analogs of the complement-inhibitingpeptide, compstatin. In particular, a compstatin analog is provided witha C-terminal and/or N-terminal modification that improves the solubilityof the peptide without causing significant reduction in itspharmacokinetic properties and, in some cases, unexpectedly conferringenhanced plasma and vitreous stability and/or binding affinity for C3and its fragments.

One aspect of the invention features a compound comprising a compstatinor compstatin analog having an amino acid sequence represented by SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6,or SEQ ID NO:7; and a terminal modification comprising an added terminalcomponent that improves (1) the peptide's C3, iC3b, C3b or C3c bindingaffinity, (2) the peptide's solubility at physiological pH, (3) thepeptide's plasma stability and/or plasma residence time, and/or (4) thepeptide's vitreous stability and/or vitreous residence time, as comparedwith an unmodified compstatin peptide under equivalent conditions.

The terminal modification can be a C-terminal component or an N-terminalcomponent. In some embodiments, the added terminal component comprisesone or more, two or more, or three or more hydrophilic/charged aminoacid residues, such as lysine, arginine, ornithine, or any combinationthereof. In a particular embodiment, the one or more hydrophilic/chargedamino acid residues are lysines.

In some embodiments, the compound comprises a compstatin analog havingan amino acid sequence represented by SEQ ID NO:7 (Cp40). In others, thecompound has an amino acid sequence represented by SEQ ID NO:8, SEQ IDNO: 9, or SEQ ID NO: 10; preferably, the amino acid sequence isrepresented by SEQ ID NO: 9 (Cp40-KK) or SEQ ID NO: 10 (Cp40-KKK).

The compound can include, additionally or alternatively, a polymercomponent. Such a component is useful to increase the bioavailability orextend the in vivo retention of the compound. In particular, theadditional component is a short polymer, typically a polyethylene glycol(PEG) having an average molecular weight of about 3 kDa or less. The PEGis bound to either the N- or C-terminus of the compstatin analog. In oneembodiment, the PEG is covalently bonded to the N-terminus via an amidelinkage. In the foregoing embodiments, the PEG can be either amonodisperse PEG having a molecular weight of about 0.5 kDa to about 3kDa or a polydisperse PEG having an average molecular weight of about0.5 kDa to about 3 kDa.

In another aspect, the compstatin peptide has the amino acid sequenceXaa1-Xaa2-Cys-Val-Xaa3-Gln-Xaa4-Xaa5-Gly-Xaa6-His-Xaa7-Cys-Xaa8 (SEQ IDNO:7), in which the Gly between Xaa5 and Xaa6 optionally is modified toconstrain the backbone conformation; wherein:

-   -   Xaa1 is absent or is Tyr, D-Tyr or Sar;    -   Xaa2 is Ile, Gly or Ac-Trp;    -   Xaa3 is Trp or an analog of Trp, wherein the analog of Trp has        increased hydrophobic character as compared with Trp;    -   Xaa4 is Asp or Asn;    -   Xaa5 is Trp or an analog of Trp comprising a chemical        modification to its indole ring wherein the chemical        modification increases the hydrogen bond potential of the indole        ring;    -   Xaa6 is His, Ala, Phe or Trp;    -   Xaa7 is Arg or Orn; and    -   Xaa8 is Thr, Ile, Leu, Nle, N-methyl Thr or N-methyl Ile,        wherein a carboxy terminal —OH of any of the Thr, Ile, Leu, Nle,        N-methyl Thr or N-methyl Ile optionally is replaced by —NH₂; and    -   the peptide is cyclic via a Cys-Cys or thioether bond.

In such aspect, the compstatin peptide includes a terminal modificationcomprising an added terminal component that improves (1) the peptide'sC3, iC3b, C3b or C3c binding affinity, (2) the peptide's solubility atphysiological pH, and/or (3) the peptide's plasma stability and/orplasma residence time; and/or (4) the peptide's vitreous stabilityand/or vitreous residence time, as compared with an unmodifiedcompstatin peptide under equivalent conditions.

In another aspect of the invention, a pharmaceutical composition isdescribed that comprises any one of the foregoing compounds combinedwith a pharmaceutically acceptable carrier. The pharmaceuticalcomposition can be formulated for a variety of administration routes,including, oral, topical, intraocular (including intravitreal or via anocular implant), periodontal (including gingival or via intrapapillaryinfiltration), pulmonary, subcutaneous, intramuscular, or intravenous.In some embodiments, a method of inhibiting complement activation isdescribed that includes the administration of any one of the foregoingpharmaceutical compositions.

In some embodiments, the compound is a compstatin or compstatin analoglinked to a polymer having an average molecular weight of about 3 kDa orless. In a particular example, the polymer is polyethylene glycol (PEG)with an average molecular weight of about 3 kDa or less. In someaspects, the PEG is linked to the N- or C-terminus. The PEG may bemonodisperse or polydisperse and may have an average molecular weight ofbetween about 0.5 kDa and 3 kDa.

In another aspect, novel Cp40 analogs are described that comprise,consist of, or consist essentially of the amino acid sequencesrepresented by SEQ ID NO:9 or SEQ ID NO:10. In some aspects, the novelCp40 analogs include Cp40-KK, Cp40-KKK, mPEG(1K)-Cp40, andmPEG(3K)-Cp40.

Also described herein are methods of treating an individual having apathological condition associated with complement activation thatinclude the steps of providing an individual having a pathologicalcondition associated with complement activation; administering to theindividual a therapeutically effective amount of any one of thepharmaceutical compositions described herein; and measuring one or moreparameters of the pathological condition. In these methods,administering of the pharmaceutical composition results in inhibition ofcomplement. The pathological condition that can be treated by thesemethods includes, but is not limited to, atypical hemolytic uremicsyndrome (aHUS); dense deposit disease (DDD); C3 glomerulonephritis(C3GN); C3 glomerulopathies; other complement-mediated nephropathies andglomerular inflammatory diseases; age-related macular degeneration(AMD); any eye disorder characterized by macular degeneration, choroidalneovascularization (CNV); retinal Neovascularization (RNV),proliferative vitreoretinopathy, glaucoma, uveitis, ocular inflammation,or any combination of these; paroxysmal nocturnal hemoglobinuria (PNH);cold agglutinin disease (CAD); warm antibody autoimmune hemolyticanemias (wAIHAs); sickle cell disease; transplant-associated thromboticmicroangiopathies; rheumatoid arthritis (RA), systemic lupuserythematosus (SLE); several autoimmune and autoinflammatory kidneydiseases; autoimmune myocarditis; multiple sclerosis; traumatic brainand spinal cord injury; cerebral, intestinal and renalischemia-reperfusion (IR) injury; spontaneous and recurrent pregnancyloss; antiphospholipid syndrome (APS); Parkinson's disease; Alzheimer'sdisease; other neurodegenerative inflammatory conditions underpinned byaberrant synaptic remodeling, excessive microglial activity andcognitive decline; asthma; anti-nuclear cytoplasmic antigen-associatedpauci-immune vasculitis (Wegener's syndrome); non-lupus autoimmune skindiseases such as pemphigus, bullous pemphigoid, and epidermolysisbullosa; post-traumatic shock, cancer; periodontitis; gingivitis; andatherosclerosis.

In some embodiments, the method employs a pharmaceutical compositionthat is administered intravenously or subcutaneously at atherapeutically effective dose of between about 0.125 mg/kg and about 10mg/kg, or between about 0.25 mg/kg and about 5 mg/kg, or between about0.5 mg/kg and about 5 mg/kg, or between about 0.5 mg/kg and about 4mg/kg, or about 3 mg/kg. In other embodiments, the pharmaceuticalcomposition is administered intramuscularly at a therapeuticallyeffective dose of between about 0.25 mg/kg and about 50 mg/kg, orbetween about 0.25 mg/kg and about 35 mg/kg, or between about 0.25 mg/kgand about 10 mg/kg, or between about 0.25 mg/kg and about 5 mg/kg, orabout 2.5 mg/kg. In other embodiments, the pharmaceutical composition isadministered orally at a therapeutically effective dose of between about1 mg/kg and about 20 mg/kg, or between about 1 mg/kg and about 10 mg/kg,or between about 1 mg/kg and about 5 mg/kg. In yet other embodiments,the pharmaceutical composition is administered intravitreally at atherapeutically effective dose of between about 1 g and about 10 mg, orbetween about 1 g and about 2,000 g, or about 1 mg. In still others, thepharmaceutical composition is administered periodontally at atherapeutically effective dose of between about 1 g and about 1,000 g,or between about 10 g and about 200 g, or between about 20 g and about100 g, or about 25 g or 50 g. In some aspects, the periodontaladministration is by intragingival injection or intrapapillaryinfiltration. These pharmaceutical compositions may be administered as asingle dose or at regular intervals ranging from once every 12 hours toonce every 3 months (e.g., once every 2-3 days, once every 2 weeks, oronce every 3 months).

Other aspects of the method envision a pharmaceutical compositionadministered intravenously or subcutaneously to the individual as afirst therapeutically effective dose that is between about 0.125 mg/kgand about 10 mg/kg (or between about 0.5 mg/kg to about 3 mg/kg) andfollowed by further administration of the pharmaceutical composition ina second therapeutically effective maintenance dose of between about0.25 mg/kg and about 50 mg/kg if administered intramuscularly or betweenabout 1 mg/kg and about 20 mg/kg if administered orally. Moreover, thesecond therapeutically effective maintenance dose may be between about0.25 mg/kg and about 10 mg/kg or between about 0.25 mg/kg and about 5mg/kg when administered intramuscularly. Alternatively, the secondtherapeutically effective dose may be administered orally at a range ofbetween about 1 mg/kg and about 10 mg/kg or about 1 mg/kg and about 5mg/kg.

In another aspect of the invention, a method of inhibiting complementactivation in an individual is provided that includes the steps ofproviding an individual; administering to the individual atherapeutically effective amount of a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and any one of theforegoing compounds; and measuring one or more parameters of complementactivation in the individual. The pharmaceutical compositions employedin this method can include any of the formulations and dosage amountsdescribed above, and can be administered as a single dose or at regularintervals ranging from once every 12 hours to once every 3 months (e.g.,every 2-3 days, every 2 weeks, or every 3 months). The pharmaceuticalcomposition can be administered as a first therapeutically effectivedose (e.g., intravenously or subcutaneously) and then as a secondtherapeutically effective maintenance dose (e.g., intramuscularly ororally). The therapeutically effective doses that may be used with thismethod include, but are not limited to: intravenous or subcutaneous inthe ranges of between about 0.125 and about 10 mg/kg, about 0.25 mg/kgand about 5 mg/kg, about 0.5 mg/kg and about 5 mg/kg, about 0.5 mg/kgand about 4 mg/kg, or about 0.5 mg/kg and about 3 mg/kg; intramuscularlyin the ranges of between about 0.25 mg/kg and about 50 mg/kg, about 0.25mg/kg and about 35 mg/kg, about 0.25 mg/kg and about 30 mg/kg, about0.25 mg/kg and about 10 mg/kg, about 0.25 mg/kg and about 5 mg/kg; ororally in the ranges of between about 1 mg/kg and about 20 mg/kg, about1 mg/kg and about 10 mg/kg, or about 1 mg/kg and about 5 mg/kg. In suchembodiments, the second therapeutically effective maintenance dose isadministered to the individual every 2-3 days or every 2 weeks. In someembodiments, the pharmaceutical composition is administered via ocularimplants at a therapeutically effective dose of between about 100 g andabout 50 mg (e.g., between about 100 g and about 10 mg, or between about100 g and about 5 mg, or between about 100 g and about 500 g). Theocular implant may be maintained on or in the eye of the individual(e.g., a human) for a period of at least about 2 days. In otherembodiments, the ocular implant may be maintained on or in the eye for aperiod of at least about 1 week. In yet others, the ocular implant ismaintained on or in an eye for a period of at least about 1 month.

In another aspect, a method of detection of a compstatin analog in abiological sample is provided herein and includes the steps of (1)providing a biological sample that comprises a first plurality ofcompstatin analog molecules, wherein at least a portion of thecompstatin analog molecules are bound to C3 and/or its fragments C3b,iC3b, and C3c to produce a plurality of C3-bound compstatin analogmolecules; (2) heat-inactivating the biological sample to produce aheat-inactivated sample wherein compstatin molecules are dissociatedfrom their target C3 molecules; (3) providing a CM5 sensor chip to whicha second plurality of compstatin analog molecules are covalentlyattached; (4) mixing the heat-inactivated sample with a pre-determinedamount of C3/C3b/iC3b/C3c or human plasma (as a source of C3) andcontacting the mixture to the CM5 sensor chip whereby the heat-releasedcompstatin analog molecules, present in the biological sample, competewith the immobilized compstatin analog molecules for binding to C3; and(5) detecting the binding of free C3/C3b/iC3b/C3c to compstatin analogmolecules on the CM5 chip, whereby the reduction of boundC3/C3b/iC3b/C3c is proportional to the presence of compstatin analogmolecules in the heat-inactivated biological sample.

In some embodiments, the method of detection utilizes surface plasmonresonance. In other embodiments, the biological sample is a vitreoussample or plasma sample extracted from a human or non-human primate.This method may be carried out using any of the analogs described above,such as Cp40-KK, Cp40-KKK, mPEG(1K)-Cp40, or mPEG(3K)-Cp40.

Also provided herein is a method of generating highly specificantibodies for the detection of Lysine-modified or unmodified compstatinanalogs that includes the steps of: (a) immunizing a first mammal with afirst compstatin or a compstatin analog; (b) immunizing a second mammalwith a second compstatin or a compstatin analog, wherein the secondcompstatin or compstatin analog has the same amino acid sequence as thefirst compstatin or compstatin analog in (a) except with one or morelysine residues linked to the C-terminus; (c) injecting the first mammalwith the first compstatin or compstatin analog, wherein the injecting isperformed every at least two days for a period of at least 2 weeks, andwherein a first plurality of antibodies are generated; (d) injecting thesecond mammal with the second compstatin or compstatin analog, whereinthe injecting is performed every at least two days for a period of atleast 2 weeks, and wherein a second plurality of antibodies aregenerated; and (e) purifying the first plurality of antibodies and thesecond plurality of antibodies. The first and second antibodies producedby this method are capable of distinguishing an antigen of the firstcompstatin or compstatin analog from an antigen of the second compstatinor compstatin analog, respectively. In a preferred embodiment, the firstand second antibodies produced by this method are monoclonal antibodies.

Other features and advantages of the present invention will beunderstood by reference to the detailed description, drawings, andexamples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, panel a) depicts the general structure of Cp40 and the Cp40N-terminal and C-terminal modifications. FIG. 1, panel b) depicts asummary of various embodiments of the Cp40-based analogs describedherein. FIG. 1, panel c) is an excerpt of the UV-HPLC-chromatograms ofCp40 and its analogs eluting between 13.9 and 18.7 min.

FIG. 2A is a summary of the MALDI spectra of the Cp40-based analogs andshows: panel a) mPEG(3k)-Cp40, panel b) mPEG(2k)-Cp40, panel c)mPEG(1k)-Cp40, panel d) mPEG(1056)-Cp40, and panel e) mPEG(528)-Cp40;FIG. 2B is a summary of the MALDI spectra of the Cp40-based analogs andshows: panel a) Cp40, panel b) Cp40-K, panel c) Cp40-KK, and panel d)Cp40-KKK.

FIG. 3 illustrates classical pathway complement inhibition by exemplarycompstatin analogs as measured by ELISA, with Cp40 in each graph beingshown for comparison. The y-axis represents the percent inhibition ofcomplement activity, and the x-axis is the concentration of the peptide.

FIG. 4 is a kinetic analysis of Cp40 as compared to Cp40 modified at theN-terminus or C-terminus. Each SPR sensorgram is the result of theanalysis of a representative example out of at least three SPRexperiments of a single-cycle kinetic titration of an individual peptideover five concentrations (black: processed SPR data; gray: kinetic fitto 1:1 Langmuir model).

FIG. 5 profiles of the binding of exemplary Cp40-based analogs to C3b,as assessed by SPR experiments after fitting to a 1:1 Langmuir bindingmodel.

FIG. 6 is a pharmacokinetic evaluation of Cp40 and exemplary modifiedCp40 peptides in NHP. Panel a) depicts a scheme of the single-dosepeptide administration via s.c. injection into two cynomolgus monkeys attime zero (vertical arrow). Cp40 or a modified Cp40 was injected intoeach animal (2 mg/kg; 8 mg net) and blood samples were collected atvarious time points (drops). Panel b) is a pharmacokinetic profile ofCp40. Panel c) is a pharmacokinetic profile of mPEG(3k)-Cp40. Panel d)is a pharmacokinetic profile of Cp40-KK. Panel e) is a pharmacokineticprofile of Cp40-KKK as assessed by UPLC-ESI-MS analysis of the NHPplasma samples. C3 levels in each animal are shown in correspondingcolors as dotted lines.

FIG. 7, panels a) and b) show the quantification of Cp40-KK in theplasma samples of two cynomolgus monkeys, as determined by UPLC-ESI-MS.FIG. 7, panels c) and d) show the quantification of Cp40-KKK in theplasma samples of two cynomolgus monkeys, as determined by UPLC-ESI-MS.Also included are the fragments that were detected in the individualsamples and the sum of the detected fragments corresponding to the totalamount of peptide in each sample.

FIG. 8 is an excerpt of the superimposed base peak intensity (BPI)chromatograms of Cp40-KKK spiked into non-human primate (NHP) plasma ata concentration of 8 (solid line) and that of four plasma samplescollected at 5 min (long dashed line), 30 min (dotted line), 1 h (dashedand dotted line), and 2 h (short dashed line) after single-dose s.c.injection of 2 mg/kg Cp40-KKK into NHP.

FIG. 9 is a graph of the plasma concentration of Cp40-KKK in cynomolgusmonkeys following three 2 mg/kg intravenous injections every 48 hours.The x-axis represents peptide concentration (M) in NHP plasma asdetermined by mass spectrometry, and the y-axis represents time afterinjection (hours). The dotted line represents the average levels ofplasma C3.

FIG. 10 is a graph of the NHP plasma concentration of Cp40 in cynomolgusmonkeys following four subcutaneous injections of 2 mg/kg Cp40administered at 0, 8, 16, and 24 hours (panel a) or 4 mg/kg Cp40administered at 0, 12, 24, and 36 hours (panel b). The x-axis representspeptide concentration (M) in NHP plasma, and the y-axis represents timeafter injection (hours). The dotted lines represent the average levelsof plasma C3.

FIG. 11 show graphs of peptide concentration over time in cynomolgusmonkeys to which was administered a single 2 mg/kg intravenous injectionof Cp40-KK (panel a), Cp40-KKK (panel b), mPEG(1k)-Cp40 (panel c), andmPEG(3k)-Cp40 (panel d). The x-axis represents peptide concentration(nM) in NHP plasma, and the y-axis represents time after injection(hours).

FIG. 12 is a quantification of peptide cleavage fragments in the plasmaof cynomolgus monkeys following a single intravenous injection with 2mg/kg of Cp40-KK (panels a and b) or Cp40-KKK (panels c and d). Thex-axis represents peptide concentration (M) in NHP plasma, as determinedby UPLC-ESI-MS, and the y-axis represents time after injection (hours).

FIG. 13 shows the peptide concentration in NHP plasma from cynomolgusmonkeys over time following a single intramuscular injection of 100 mgof Cp40 (panel a), Cp40-KK (panel b), and Cp40-KKK (panel c). The x-axisrepresents peptide concentration (M) in NHP plasma, as determined by MS,and the y-axis represents time after injection (days).

FIG. 14 is an exemplary direct ELISA assay showing the specificity ofthe Cp40 and Cp40-KKK antibodies fixed in an ELISA plate. The Cp40antibody (AB-101) reacts with the biological sample containing the Cp40peptide (solid circle), but with 10-fold lower titer to Cp40-KKK peptide(solid square). Likewise, the Cp40-KKK antibody (AB-102) reacts with thebiological sample containing the Cp40-KKK peptide (unshaded square), butwith 10-fold lower titer to Cp40 peptide (unshaded circle). The y-axisrepresents the OD reading at 405 nm, whereas the x-axis represents theantiserum dilution.

FIG. 15 is a representative five-point standard curve (panel a) andsimple western blot gel (panel b) obtained upon running predeterminedamounts of Cp40-KKK peptide spiked into rabbit vitreous samples. Inpanel a), the x-axis represents peptide concentration (nM), and they-axis represents peak area.

FIG. 16 represents a schematic diagram of the SPR method forquantitation of the Cp40 analogs in biological fluids. Plasma sampleswere diluted and heated at 95° C. for 5 m (#3) followed bycentrifugation and addition of C3 or a plasma source of C3 (#4). Thesamples were then flown over the corresponding Cp40 analog-immobilizedchip (#5).

FIG. 17 is a representative standard curve obtained via surface plasmonresonance analysis of rabbit vitreous samples spiked with predeterminedamounts of Cp40-KKK peptide. The x-axis represents peptide concentration(nM), and the y-axis represents relative light units.

FIG. 18 depicts the concentration of mPEG(3k)-Cp40 (dotted bar),mPEG(1k)-Cp40 (checkered bar), Cp40-KK (black bar), and Cp40-KKK (whitebar) compounds detected in the vitreous samples from cynomolgus monkeysafter a single intravitreal injection. The x-axis represents the numberof days following injection with 500 g of compound, and the y-axisrepresents the concentration of compound (nM).

FIG. 19 is an SPR profile of Cp40-KKK binding to purified C3b. Panel a)is a positive control, standard curve showing C3b binding by Cp40-KKKadded to rabbit vitreous at concentrations of 2, 4, 8, and 16 nM. Thex-axis represents peptide concentration (nM), and the y-axis representsrelative light units. Panel b) shows the C3b binding signal obtainedfrom NHP eye vitreous injected with Cp40-KKK. The y-axis representsrelative light units. The vitreous samples were either subjected to heatinactivation (white bar) or not subjected to heat inactivation (blackbar) prior to chip immobilization.

FIG. 20 depicts classical complement pathway inhibition in human eyeplasma incubated with Cp40 (panel a), Cp40-K (panel b), Cp40-KK (panelc), and Cp40-KKK (panel d) in the presence of rabbit vitreous (square)or absence of rabbit vitreous (circle). The x-axis represents analogconcentration (nM), and the y-axis represents percent inhibition of C3bdeposition.

FIG. 21 is a summary of the in vivo and in vitro Lys-cleavage in Cp40-KK(panel a) and Cp40-KKK (panel b).

DETAILED DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS Definitions

Various terms relating to the methods and other aspects of the presentinvention are used throughout the specification and claims. Such termsare to be given their ordinary meaning in the art unless otherwiseindicated. Other specifically defined terms are to be construed in amanner consistent with the definition provided herein.

The following abbreviations may be used herein: Ac, acetyl group; BSA,bovine serum albumin; DCM, dichloromethane; DMF, dimethylformamide;ELISA, enzyme-linked immunosorbent assay; ESI, electrospray ionization;Fmoc, 9-fluorenylmethoxycarbonyl; MALDI-TOF-MS, matrix-assisted laserdesorption ionization-time-of-flight mass spectrometry; NHP, non-humanprimate; PBS, Phosphate Buffered Saline; RP-HPCL, reversed-phasehigh-performance liquid chromatography; Sar, N-methyl glycine; s.c.,subcutaneous; SPR, surface plasmon resonance; TFA, trifluoroacetic acid;UPLC-ESI-MS, ultra-performance liquid chromatography-electrosprayionization-tandem mass spectrometry; VBS, Veronal buffered saline; WFI,water for injection.

The singular form of a word includes the plural, and vice versa, unlessthe context clearly dictates otherwise. The, the references “a”, “an”,and “the” are generally inclusive of the plurals of the respectiveterms. For example, references to “a compound” or “a method” includes aplurality of such “compounds” or “methods.” Similarly, the words“comprise”, “comprises”, and “comprising” are to be interpretedinclusively rather than exclusively. Likewise, the terms “include”,“including”, and “or” should all be construed to be inclusive, unlesssuch a construction is clearly prohibited from the context.

The terms “comprising” or “including” are intended to includeembodiments encompassed by the terms “consisting essentially of” and“consisting of” Similarly, the term “consisting essentially of” isintended to include embodiments encompassed by the term “consisting of.”Moreover, the term “consisting essentially of” limits the scope of anembodiment to the specified components or steps and those components orsteps that do not materially affect the basic and novel characteristicsof the embodiment.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or ±10%, in some embodiments ±5%, in someembodiments ±1%, and in some embodiments ±0.1% from the specified value,as such variations are appropriate to make and use the disclosedcompounds and compositions.

The term “compstatin” as used herein refers to a peptide comprising SEQID NO:1, I[CVVQDWGHHRC]T (cyclic C2-C12 by way of a disulfide bondindicated by the brackets). The term “compstatin analog” refers to amodified compstatin comprising substitutions of natural and/or unnaturalamino acids, or amino acid analogs, as well as modifications within orbetween various amino acids, as described in greater detail herein, andas known in the art. When referring to the location of particular aminoacids or analogs within compstatin or compstatin analogs, thoselocations are sometimes referred to as “positions” within the peptide,with the positions numbered from 1 (Ile in compstatin) to 13 (Thr incompstatin). For example, the Gly residue occupies “position 8.”

The terms “pharmaceutically active” and “biologically active” refer tothe ability of the compounds of the invention to bind C3 or fragmentsthereof and inhibit complement activation. This biological activity maybe measured by one or more of several art-recognized assays, asdescribed in greater detail herein.

As used herein, “alkyl” refers to an optionally substituted saturatedstraight, branched, or cyclic hydrocarbon having from about 1 to about10 carbon atoms (and all combinations and subcombinations of ranges andspecific numbers of carbon atoms therein), with from about 1 to about 7carbon atoms being preferred. Alkyl groups include, but are not limitedto, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl,n-pentyl, cyclopentyl, isopentyl, neopentyl, n-hexyl, isohexyl,cyclohexyl, cyclooctyl, adamantyl, 3-methylpentyl, 2,2-dimethylbutyl,and 2,3-dimethylbutyl. The term “lower alkyl” refers to an optionallysubstituted saturated straight, branched, or cyclic hydrocarbon havingfrom about 1 to about 5 carbon atoms (and all combinations andsubcombinations of ranges and specific numbers of carbon atoms therein).Lower alkyl groups include, but are not limited to, methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, cyclopentyl,isopentyl, and neopentyl.

As used herein, “halo” refers to F, Cl, Br, or I.

As used herein, “alkanoyl”, which may be used interchangeably with“acyl”, refers to an optionally substituted straight or branchedaliphatic acylic residue having from about 1 to about 10 carbon atoms(and all combinations and subcombinations of ranges and specific numbersof carbon atoms therein), with from about 1 to about 7 carbon atomsbeing preferred. Alkanoyl groups include, but are not limited to,formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, isopentanoyl,2-methyl-butyryl, 2,2-dimethylpropionyl, hexanoyl, heptanoyl, octanoyl,and the like. The term “lower alkanoyl” refers to an optionallysubstituted straight or branched aliphatic acylic residue having fromabout 1 to about 5 carbon atoms (and all combinations andsubcombinations of ranges and specific numbers of carbon atoms therein.Lower alkanoyl groups include, but are not limited to, formyl, acetyl,n-propionyl, iso-propionyl, butyryl, isobutyryl, pentanoyl,iso-pentanoyl, and the like.

As used herein, “aryl” refers to an optionally substituted, mono- orbicyclic aromatic ring system having from about 5 to about 14 carbonatoms (and all combinations and subcombinations of ranges and specificnumbers of carbon atoms therein), with from about 6 to about 10 carbonsbeing preferred. Non-limiting examples include, for example, phenyl andnaphthyl.

As used herein, “aralkyl” refers to alkyl as defined above, bearing anaryl substituent and having from about 6 to about 20 carbon atoms (andall combinations and subcombinations of ranges and specific numbers ofcarbon atoms therein), with from about 6 to about 12 carbon atoms beingpreferred. Aralkyl groups can be optionally substituted. Non-limitingexamples include, for example, benzyl, naphthylmethyl, diphenylmethyl,triphenylmethyl, phenylethyl, and diphenylethyl.

As used herein, the terms “alkoxy” and “alkoxyl” refer to an optionallysubstituted alkyl-O— group wherein alkyl is as previously defined.Exemplary alkoxy and alkoxyl groups include methoxy, ethoxy, n-propoxy,i-propoxy, n-butoxy, and heptoxy, among others.

As used herein, “carboxy” refers to a —C(═O)OH group.

As used herein, “alkoxycarbonyl” refers to a —C(═O)O-alkyl group, wherealkyl is as previously defined.

As used herein, “aroyl” refers to a —C(═O)-aryl group, wherein aryl isas previously defined. Exemplary aroyl groups include benzoyl andnaphthoyl.

Typically, substituted chemical moieties include one or moresubstituents that replace hydrogen at selected locations on a molecule.Exemplary substituents include, for example, halo, alkyl, cycloalkyl,aralkyl, aryl, sulfhydryl, hydroxyl (—OH), alkoxyl, cyano (—CN),carboxyl (—COOH), acyl (alkanoyl: —C(═O)R); —C(═O)O-alkyl, aminocarbonyl(—C(═O)NH₂), —N— substituted aminocarbonyl (—C(═O)NHR″), CF₃, CF₂CF₃,and the like. In relation to the aforementioned substituents, eachmoiety R″ can be, independently, any of H, alkyl, cycloalkyl, aryl, oraralkyl, for example.

As used herein, “L-amino acid” refers to any of the naturally occurringlevorotatory alpha-amino acids normally present in proteins or the alkylesters of those alpha-amino acids. The term “D-amino acid” refers todextrorotatory alpha-amino acids. Unless specified otherwise, all aminoacids referred to herein are L-amino acids.

“Hydrophobic” or “nonpolar” are used synonymously herein, and refer toany inter- or intra-molecular interaction not characterized by a dipole.

“PEGylation” refers to the reaction in which at least one polyethyleneglycol (PEG) moiety, regardless of size, is chemically attached to aprotein or peptide to form a PEG-peptide conjugate. “PEGylated” meansthat at least one PEG moiety, regardless of size, is chemically attachedto a peptide or protein. The term PEG is generally accompanied by anumeric suffix that indicates the approximate average molecular weightof the PEG polymers; for example, PEG-8,000 refers to polyethyleneglycol having an average molecular weight of about 8,000 Daltons (org/mol).

“Monodisperse” refers to a polymer composed of molecules having chainlengths of approximately the same mass.

“Polydisperse” refers to a polymer composed of molecules having chainlengths over a range of molecular masses, where the mass is typicallyindicated by the average molecular weight.

As used herein, “pharmaceutically acceptable salts” or “pharmaceuticallyacceptable esters” refer to derivatives of the disclosed compoundswherein the parent compound is modified by making an ester or an acid orbase salt form, which is compatible with any other ingredients of thepharmaceutical composition, and which is not deleterious to the subjectto which the composition is to be administered. Examples ofpharmaceutically-acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. Thus, the term “acid addition salt” refers to the correspondingsalt derivative of a parent compound that has been prepared by theaddition of an acid. The pharmaceutically-acceptable salts include theconventional salts or the quaternary ammonium salts of the parentcompound formed, for example, from inorganic or organic acids. Forexample, such conventional salts include, but are not limited to, thosederived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfamic, phosphoric, nitric, and the like; and the saltsprepared from organic acids such as acetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic,ethane disulfonic, oxalic, isethionic, and the like. Certain acidic orbasic compounds of the present invention may exist as zwitterions. Allforms of the compounds, including free acid, free base, and zwitterions,are contemplated to be within the scope of the present invention.

As used herein, the phrase “pharmaceutically suitable fluids or“pharmaceutically suitable liquids,” especially with reference tosolubility of the compounds of the invention, but not limited thereto,refers collectively to fluids that include but are not limited tobuffers and other aqueous solutions having a physiological pH, as wellas non-aqueous solvents and liquid media commonly used for thepreparation and delivery of drugs to the body by various routes asdiscussed herein. Such non-aqueous solvents and liquid media include:polar protic and/or aprotic non-aqueous organic solvents such as loweralcohols, methyl and vinyl pyrrolidones such as polyvinylpyrrolidone,methylsulfonyl methane, dimethylsulfoxide and related compounds, hydroxyand polyhydroxy acids such as polylactic acid, among others. This phrasemay be used interchangeably with the term “clinically relevantsolvents.”

As used herein, the term “pharmaceutically-acceptable carrier” means achemical composition with which a compstatin analog may be combined andwhich, following the combination, can be used to administer thecompstatin analog to an individual.

As used herein, “intraocular administration” or “ocular administration”of a pharmaceutical composition includes any route of administrationcharacterized by introduction into the eye, including “intravitrealadministration.” The term “intravitreal administration” of apharmaceutical composition includes any route of administrationcharacterized by introduction into the vitreous cavity of the eye. The“vitreous” is a gel-like substance within the vitreous cavity that fillsthe space between the lens and the retina and helps the eye maintain itsshape.

As used herein, “intramuscular administration” of a pharmaceuticalcomposition includes any route of administration characterized byintroduction into the muscles.

As used herein, “periodontal administration” of a pharmaceuticalcomposition refers to the administration within the tissues surroundingand/or around a tooth or teeth (e.g., by injection, topical application,or biodegradable implant), and includes “gingival administration” and“intrapapillary infiltration.” As used herein, “gingival administration”of a pharmaceutical composition refers includes any route ofadministration characterized by introduction to or into the gingiva, orgums. “Intrapapillary infiltration” or “intrapapillary infiltrationinjection” is a type of gingival administration that refers toadministration of a pharmaceutical composition into the interdentalpapilla, which is the gingiva (gum) tissue that exists coronal to thefree gingival margin on the buccal and lingual surfaces of the teeth.

As used herein, “oral administration” or “enteral administration” of apharmaceutical composition includes any route of administrationcharacterized by introduction into the gastrointestinal tract. “Oraladministration” includes feeding by mouth as well as orogastric orintragastric gavage. “Oral administration” or “enteral administration”also may include sublingual, buccal, intranasal, pulmonary or rectaladministration, among other routes known in the art.

As will be appreciated by the skilled artisan, “physiological pH”typically refers to the pH of human blood, which is maintained between7.35 and 7.45. As used herein, the term “physiological pH includes a pHrange of 7.3 to 7.5.

The term “treating” refers to any indicia of success in the treatment oramelioration of the disease or condition. Treating can include, forexample, reducing or alleviating the severity of one or more symptoms ofthe disease or condition, or it can include reducing the frequency withwhich symptoms of a disease, defect, disorder, or adverse condition, andthe like, are experienced by an individual, such as a human patient.

The term “preventing” refers to the prevention of the disease orcondition in an individual, such as a human patient. For example, if anindividual at risk of developing an inflammatory disease is treated withthe compounds and/or using the methods of the present invention and doesnot later develop the disease or condition, then the disease has beenprevented in that individual.

The term “treat or prevent” is sometimes used herein to refer to amethod that results in some level of treatment or amelioration of thedisease or condition, and contemplates a range of results directed tothat end, including but not restricted to prevention of the conditionentirely.

The term “parameter” as used herein to refer to measuring any bodilyfunction that is observable or measurable using suitable measuringtechniques available in the art. As one having ordinary skill in the artwill appreciate, measuring one or more “parameters” of bodily functioncan be used to detect a particular dysfunction as compared to theaverage normal parameters and can also be used to determine whether thatbodily function has improved following or during treatment. Suchparameters can be general, e.g., body temperature, blood pressure, pulse(heart rate), and breathing rate (respiratory rate), or they can bespecific to a particular organ, tissue or disease or condition, e.g.,functional test results from blood or other organs/tissues.

The terms “therapeutically effective amount” or “therapeuticallyeffective dose” is the amount of a pharmaceutical composition sufficientto provide a beneficial effect to the individual to whom thepharmaceutical composition is administered.

Description:

The present invention arises in part from the inventors' development ofcompstatin analogs displaying increased solubility and improvedpharmacokinetic parameters. Modification of the compstatin or compstatinanalog with small polymers (e.g., about 3,000 Da or less) at theN-terminus results in compstatin analogs with improved solubility inclinically relevant solvents while, at the same time, having complementinhibitory activity similar to that of the unmodified parent compoundunder equivalent conditions. Polydisperse, and particularlymonodisperse, polyethylene glycol (PEG) of average molecular weight˜500-3,000 Da are particularly suitable.

Additionally, modification of the compstatin or compstatin analog at theN-terminus or C-terminus with the addition of one or more chargedhydrophilic amino acid residues, such as lysine, can confer improvedpharmacokinetic properties (e.g., increased solubility) to thecompstatin or compstatin analog. For instance, described herein aremodifications of the compstatin or compstatin analog at the C-terminuswith the addition of one or more charged hydrophilic residues (e.g.,lysine), which not only increases the solubility of the peptide, butunexpectedly enhances the residence time and binding affinity of thecompstatin analog for C3 or its fragments. Pharmacokinetic evaluation ofthe modified compstatin analog Cp40 in non-human primates revealedplasma half-life values for the modified Cp40 peptides similar to, oreven exceeding, that of the unmodified Cp40-based analog. Thus, thepresent compstatin analogs exhibit improved pharmacokinetic profiles aswell as improved solubility at physiological pH.

Thus, one modification in accordance with the present inventioncomprises adding a component to the C-terminus of compstatin(Ile-Cys-Val-Val-Gln-Asp-Trp-Gly-His-His-Arg-Cys-Thr (cyclic C2-C12)(SEQ ID NO:1), or any analog thereof as described in more detail below,that improves solubility of the peptide at physiological pH, whilemaintaining a similar C3 binding affinity, plasma half-life, and/orcomplement inhibitory activity as compared to the unmodified parentpeptide under equivalent conditions. For instance, in some embodiments,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more hydrophilic and/or charged aminoacids (e.g., Arg or Lys) are added to the C-terminus. In someembodiments, two or more hydrophilic and/or charged amino acid residuesare added to the C-terminus of the compstatin analog. In otherembodiments, three or more hydrophilic/charged amino acid residues areadded to the C-terminus of the compstatin analog. In particularembodiments, the hydrophilic/charged amino acid residue is Lys, Arg or acombination thereof. In a more preferred embodiment, one or more lysineamino acid residues are added to the C-terminus of compstatin or acompstatin analog. For instance, in an exemplary embodiment described inmore detail below, one or more lysine amino acid residues are added tothe C-terminus of the compstatin analog Cp40.

Exemplary embodiments of the invention feature the compstatin analogCp40, in which two or more lysine amino acid residues are added to theC-terminus. As described in greater detail below and in the examples,the inventors have discovered that Cp40-KK and Cp40-KKK not only exhibitincreased solubility as compared to unmodified Cp40, but, surprisingly,exhibit increased plasma and vitreous retention and enhanced C3-bindingas compared to Cp40 and other Cp40-based analogs. In fact, Cp40-KKKdisplays in vivo residence times equal to or exceeding three monthsfollowing intravitreal administration. Notably, both Cp40-KK andCp40-KKK analogs display markedly improved pharmacokinetic properties ascompared to Cp40.

Another modification in accordance with the present invention comprisesadding a component to either or both of the N- or C-termini ofcompstatin or analogs thereof that improves solubility of the peptide atphysiological pH, while maintaining a similar C3 binding affinity,plasma half-life, and/or complement inhibitory activity as compared tothe unmodified parent peptide under equivalent conditions. In particularembodiments, the added component is the addition of a short polymer,e.g., polyethylene glycol (PEG) with shorter chain length than PEGs thathave been used previously. PEG used in accordance with the presentinvention have an average molecular weight of about 500 to about 5,000,e.g., 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500,1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500,2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500,3,600, 3,700, 3,800, 3,900, 4,00, 4,100, 4,200, 4,300, 4,400, 4,500,4,600, 4,700, 4,800, 4,900, or 5,000. In preferred embodiments, the PEGshave an average molecular weight of less than 5,000. In one embodiment,the compstatin analog is modified at one or both termini to include PEGhaving an average molecular weight of about 500 to about 5,000 Da. Inanother embodiment, the PEG has an average molecular weight of about1,000 to about 3,000 Da. In an exemplary embodiment, the Cp40-basedanalog is modified to include an N-terminal PEG with an averagemolecular weight of about 1,000 to about 3,000 Da.

The polymer modification may be a monodisperse PEG or a polydisperse PEGcovalently bonded to compstatin or a compstatin analog. For instance, inone embodiment, the terminal modification is a monodisperse PEG having amolecular weight of about 500 to about 1,000. In other embodiments, themodification is a polydisperse PEG having an average molecular weight ofabout 1,000 to about 3,000. Monodisperse PEG is particularly suitablefor use in the present invention because it facilitates purification ofthe compounds to homogeneity by enabling collection of the PEGylatedcompounds from a substantially single peak (see, e.g., FIG. 2A, panelsa-c as compared with panels d and e).

The compstatin and compstatin analogs of the present invention can becovalently bonded to PEG via a linking group. Such methods are wellknown in the art. (Reviewed in Kozlowski A. et al., 2001, BioDrugs NM:419-29; see also, Harris J M and Zalipsky S, eds. Poly(ethylene glycol),Chemistry and Biological Applications, ACS Symposium Series 680 (1997)).Non-limiting examples of acceptable linking groups include an estergroup, an amide group, an imide group, a carbamate group, a carboxylgroup, a hydroxyl group, a carbohydrate, a succinimide group (includingwithout limitation, succinimidyl succinate (SS), succinimidyl propionate(SPA), succinimidyl carboxymethylate (SCM), succinimidyl succinamide(SSA) and N-hydroxy succinimide (NHS)), an epoxide group, anoxycarbonylimidazole group (including without limitation,carbonyldimidazole (CDI)), a nitro phenyl group (including withoutlimitation, nitrophenyl carbonate (NPC) or trichlorophenyl carbonate(TPC)), a trysylate group, an aldehyde group, an isocyanate group, avinylsulfone group, a tyrosine group, a cysteine group, a histidinegroup or a primary amine. In certain embodiments, the linking group is asuccinimide group. In one embodiment, the linking group is NHS.

The compstatin and compstatin analogs of the present invention canalternatively be coupled directly to PEG (i.e., without a linking group)through an amino group, a sulfhydryl group, a hydroxyl group or acarboxyl group. In one embodiment, PEG is coupled to a lysine residueadded to the C-terminus of compstatin. In a particular embodiment, PEGis coupled to the compstatin or compstatin analog N-terminus via anamide linkage.

In some embodiments, a modification to compstatin or a compstatin analogcomprises both PEGylation and adding one or more hydrophilic or chargedamino acid residues to the C-terminus. In certain of these embodiments,PEG is coupled directed to the compstatin or compstatin analogN-terminus via an amide linkage and the PEG has an average molecularweight of about 500 to about 3,000, or an actual molecular weight inthat range, if monodisperse. In certain of these embodiments, one ormore lysine residues is covalently linked to the C-terminus.

In exemplary embodiments utilizing PEG (mw 500-3,000 DA) as the polymerand lysine as the charged residue, various arrangements of componentscan be selected as follows (wherein “Comp A” stands for “compstatinanalog”):

PEG-Comp A

-   -   Comp A-Lys₍₁₋₃₎

PEG-Comp A-Lys₍₁₋₃₎

-   -   Comp A-Lys₍₁₋₃₎-PEG

PEG-Comp A-Lys₍₁₋₃₎-PEG

The molecular weights of these modified compstatin analogs can beincreased or decreased by changing the size of the PEG. The variety ofcommercially available PEGs will also be appreciated by the skilledperson, including polydisperse PEG, monodisperse PEG, heterobifunctionaland branched PEG (see, e.g., Creative PEGWorks, Chapel Hill, N.C.;XL-Protein GMBH, Freising, Germany; BroadPharm, San Diego, Calif.)

If the N-terminus of the compstatin analog is not modified with apolymer (i.e. the arrangement in the exemplary series above is CompA-Lys₍₁₋₃₎ or Comp A-Lys₍₁₋₃₎-PEG), then the N-terminus can be modifiedin another way. For instance, an albumin-binding small molecule (e.g.ABM2) can be linked to the N-terminus. A particular embodiment of thistype features the compstatin analog Cp40 comprising 1-3 charged residues(e.g., Lys) linked to the C-terminus and ABM2 linked to the N-terminusvia an amide linkage.

The N-terminal and/or C-terminal modifications described herein increasethe solubility of the compstatin or compstatin analog in fluids at a pHof about 7.3 to about 7.5 as compared to the unmodified peptide underequivalent conditions. In certain embodiments, the increase insolubility is at least about 5-fold. In a particular embodiment, theincrease in solubility is at least about 10-fold, or at least about20-fold, or at least about 30-fold, or at least about 40-fold, or atleast about 50-fold, or at least 100-fold, 150-fold, 200-fold, 300-fold,400-fold, or even 500-fold or more, as compared to the equivalent, butunmodified peptide under equivalent conditions. Further, in particularaspects, the compstatin or compstatin analogs containing the N-terminaland/or C-terminal modifications described herein exhibit less than abouta 2-fold decrease in complement inhibitory activity as compared to theunmodified peptide under equivalent conditions. Additionally, inpreferred embodiments, the compstatin or compstatin analogs containingthe N-terminal and/or C-terminal modifications described herein exhibitless than about a 5-fold decrease in C3 binding affinity as compared tothe unmodified peptide under equivalent conditions. In some aspects, thecompstatin or compstatin analogs containing the N-terminal and/orC-terminal modifications described herein exhibit less than about a10-fold decrease in C3 binding affinity as compared to the unmodifiedpeptide under equivalent conditions. In other aspects, the compstatin orcompstatin analogs containing the N-terminal and/or C-terminalmodifications described herein exhibit an increase in C3 bindingaffinity as compared to the unmodified peptide under equivalentconditions.

As an exemplary illustration, whereas the Cp40 N-terminal modificationby conjugation of mPEG resulted in a ˜3- to 6-fold decrease in theassociation constant (k_(a)) with its ligand, C3, no significantvariation was observed in the dissociation constant (k_(d)) whencompared to the parental compound, Cp40. The lower k_(a) values werereflected in lower affinity values, especially for the analogs carryinglarger PEG chains (mPEG(3k)-Cp40, 7.9 nM; mPEG(2k)-Cp40, 4.4 nM), whencompared with Cp40 (K_(D), 0.5 nM). The apparent lower affinity,however, did not significantly affect the complement inhibitory activityof the analogs (see Table 2 in the Examples). Similarly, the addition ofLys residues to Cp40 did not significantly influence any of thebiochemical parameters mentioned above, indicating that the chosenmodifications did not induce major changes in the interaction betweenthe analogs and their ligand, C3 (Table 2). It is emphasized here thatthe dissociation Kd is of greatest significance to the residence time ofthe compound.

The C- and/or N-terminal modifications described herein can be appliedto compstatin itself or any analog thereof. Non-limiting examples ofcompstatin analogs suitable for use with the N-terminal and/orC-terminal modifications disclosed herein will now be described infurther detail.

Compstatin Analogs

The above-described N-terminal and C-terminal modifications can becombined with other modifications of compstatin previously shown toimprove activity, thereby producing peptides with significantly improvedcomplement inhibitory activity. For example, in embodiments wherein theN-terminus is not PEGylated, the N-terminus can be acetylated.Additionally, it is known that substitution of Ala for His at position 9improves activity of compstatin and is a preferred modification of thepeptides of the present invention as well.

It was disclosed in WO2004/026328 and WO2007/062249 that Trp and certainTrp analogs at position 4, as well as certain Trp analogs at position 7,especially combined with Ala at position 9, yields many-fold greateractivity than that of compstatin. These modifications are used toadvantage in the present invention as well.

In particular, peptides comprising 5-fluoro-tryptophan or either5-methoxy-, 5-methyl- or 1-methyl-tryptophan, or 1-formyl-tryptophan atposition 4 have been shown to possess many-fold greater activity thanunmodified compstatin. Particularly preferred are 1-methyl and 1-formyltryptophan. It is believed that an indole ‘N’-mediated hydrogen bond isnot necessary at position 4 for the binding and activity of compstatin.The absence of this hydrogen bond or reduction of the polar character byreplacing hydrogen with lower alkyl, alkanoyl or indole nitrogen atposition 4 enhances the binding and activity of compstatin. In certainembodiments, Trp at position 4 of compstatin is replaced with an analogcomprising a 1-alkyl substituent, more particularly a lower alkyl (e.g.,C₁-C₅) substituent as defined above. These include, but are not limitedto, N( )methyl tryptophan and 5-methyltryptophan. In other embodiments,Trp at position 4 of compstatin is replaced with an analog comprising a1-alkanoyl substituent, more particularly a lower alkanoyl (e.g., C₁-C₅)substituent as defined above, e.g., N( )formyl tryptophan,1-acetyl-L-tryptophan and L-homotryptophan.

It was disclosed in WO2007/062249 that incorporation of5-fluoro-tryptophan at position 7 in compstatin increased the enthalpyof the interaction between the resulting compstatin analog and C3,relative to compstatin, whereas incorporation of 5-fluoro-tryptophan atposition 4 in decreased the enthalpy of this interaction. Accordingly,modifications of Trp at position 7, as described in WO2007/062249, arecontemplated as useful modifications in combination with the N-terminalmodifications described above.

An exemplary compstatin analog described in WO2007/062249 is:Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa3-Gly-Xaa4-His-Arg-Cys-Xaa5 (cyclic C2-C12)(SEQ ID NO:2); wherein:

Xaa1 is Ile, Val, Leu, Ac-Ile, Ac-Val, Ac-Leu or a dipeptide comprisingGly-Ile;

Xaa2 is Trp or an analog of Trp, wherein the analog of Trp has increasedhydrophobic character as compared with Trp, with the proviso that, ifXaa3 is Trp, Xaa2 is the analog of Trp;

Xaa3 is Trp or an analog of Trp comprising a chemical modification toits indole ring wherein the chemical modification increases the hydrogenbond potential of the indole ring;

Xaa4 is His, Ala, Phe or Trp;

Xaa5 is L-Thr, D-Thr, Ile, Val, Gly, a dipeptide comprising Thr-Asn, ora dipeptide comprising Thr-Ala, or a tripeptide comprising Thr-Ala-Asn,wherein a carboxy terminal —OH of any of the L-Thr, D-Thr, Ile, Val, Glyor Asn optionally is replaced by —NH₂; and

the two Cys residues are joined by a disulfide bond.

In various embodiments, the analog of Trp of Xaa2 is a halogenated Trp,such as 5-fluoro-1-tryptophan or 6-fluoro-1-tryptophan. In otherembodiments, the Trp analog at Xaa2 comprises a lower alkoxy or loweralkyl substituent at the 5 position, e.g., 5-methoxytryptophan or5-methyltryptophan. In other embodiments, the Trp analog at Xaa 2comprises a lower alkyl or a lower alkanoyl substituent at the 1position, with exemplary embodiments comprising 1-methyltryptophan or1-formyltryptophan. In other embodiments, the analog of Trp of Xaa3 is ahalogenated Trp such as 5-fluoro-1-tryptophan or 6-fluoro-1-tryptophan.

In certain embodiments, Xaa2 comprises a lower alkanoyl or lower alkylsubstituent at the 1 position of tryptophan, Xaa3 optionally comprises ahalogenated tryptophan and Xaa4 comprises Ala.

Other class compstatin analogs with various modifications to SEQ ID Nos:1 and 2 is described in WO2010/127336. One modification disclosed inthat document comprises constraint of the peptide backbone at position 8of the peptide. In a particular embodiment, the backbone is constrainedby replacing glycine at position 8 (Gly⁸) with N methyl glycine. Anothermodification disclosed in that document comprises replacing Thr atposition 13 with Ile, Leu, Nle (norleucine), N methyl Thr or N methylIle. This class of analogs is represented by the sequence:

Xaa1-Cys-Val-Xaa2-Gln-Asp-Xaa3-Gly-Xaa4-His-Arg-Cys-Xaa5

(cyclic C2-C12) (SEQ ID NO:3) in which Gly at position 8 is modified toconstrain the backbone conformation of the peptide at that location, andwherein:Xaa1 is Ile, Val, Leu, Ac-Ile, Ac-Val, Ac-Leu or a dipeptide comprisingGly-Ile;Xaa2 is Trp or an analog of Trp, wherein the analog of Trp has increasedhydrophobic character as compared with Trp;Xaa3 is Trp, or an analog of Trp comprising a chemical modification toits indole ring wherein the chemical modification increases the hydrogenbond potential of the indole ring;

Xaa4 is His, Ala, Phe or Trp; and

Xaa5 is Thr, Ile, Leu, Nle, N-methyl Thr or N-methyl Ile, wherein acarboxy terminal —OH of any of the Thr, Ile, Leu, Nle, N-methyl Thr orN-methyl Ile optionally is replaced by —NH₂.

In embodiments of this class of analog, the Trp analog at Xaa2 comprisesa lower alkoxy or lower alkyl substituent at the 5 position, e.g.,5-methoxytryptophan or 5-methyltryptophan; or a lower alkyl or a loweralkanoyl substituent at the 1 position, with exemplary embodimentscomprising 1-methyltryptophan or 1-formyltryptophan. In otherembodiments, the analog of Trp of Xaa3 is a halogenated tryptophan suchas 5-fluoro-1-tryptophan or 6-fluoro-1-tryptophan.

In certain embodiments of this class of analog, the Gly at position 8 isN-methylated, and Xaa1 is Ac-Ile, Xaa2 is 1-methyl-Trp or 1-formyl-Trp,Xaa3 is Trp, Xaa4 is Ala, and Xaa5 is Thr, Ile, Leu, Nle, N-methyl Thror N-methyl Ile. In particular, Xaa5 may be Ile, N-methyl Thr orN-methyl Ile. In particular, the compstatin analog comprises the analogCp20: Ac-Ile-[Cys-Val-Trp(Me)-Gln-Asp-Trp-Sar-Ala-His-Arg-Cys]-mIle-NH₂(SEQ ID NO:4)

Another type of modification to compstatin analogs is described inWO2012/040259. One such modification comprises replacement of the C2-C12disulfide bond with addition of a CH₂ to form a homocysteine at C2 orC12, and introduction of a thioether bond, to form a cystathionine, suchas a gamma-cystathionine or a delta-cystathionine. Another modificationcomprises replacement of the C2-C12 disulfide bond with a thioether bondwithout the addition of a CH₂, thereby forming a lantithionine. Theanalogs comprising the thioether bond demonstrate activity that issubstantially the same as that of certain of the disulfide bond analogsand also possess equivalent or improved stability characteristics.

Another class of compstatin analogs particularly suitable as peptidescaffolds for the C-terminal or N-terminal modifications of the presentinvention in order to produce novel compounds with new pharmacokineticproperties is described in WO2013/036778. This class of analogs isrepresented by the amino acid sequenceXaa1-Xaa2-Cys-Val-Xaa3-Gln-Xaa4-Xaa5-Gly-Xaa6-His-Xaa7-Cys-Xaa8 (SEQ IDNO: 5), in which the Gly between Xaa5 and Xaa6 optionally is modified toconstrain the backbone conformation;

wherein Xaa1 is absent or is Tyr, D-Tyr or Sar;

Xaa2 is Ile, Gly or Ac-Trp;

Xaa3 is Trp or an analog of Trp, wherein the analog of Trp has increasedhydrophobic character as compared with Trp;

Xaa4 is an Asp or Asn;

Xaa5 is Trp or an analog of Trp comprising a chemical modification toits indole ring wherein the chemical modification increases the hydrogenbond potential of the indole ring;

Xaa6 is His, Ala, Phe or Trp;

Xaa7 is Arg or Orn; and

Xaa8 is Thr, Ile, Leu, Nle, N-methyl Thr or N-methyl Ile, wherein acarboxy terminal —OH of any of the Thr, Ile, Leu, Nle, or N-methyl Thror N-methyl Ile optionally is replaced by —NH₂, and the peptide iscyclic via a Cys-Cys or thioether bond.

The analog of Trp of Xaa3 may be a halogenated tryptophan, such as5-fluoro-1-tryptophan or 6-fluoro-1-tryptophan. The Trp analog at Xaa3may comprise a lower alkoxy or lower alkyl substituent at the 5position, e.g., 5-methoxy-tryptophan or 5-methyltryptophan. In otherembodiments, the Trp analog at Xaa3 comprises a lower alkyl or a loweralkanoyl substituent at the 1 position, with exemplary embodimentscomprising 1-methyltryptophan or 1-formyl-tryptophan. In otherembodiments, the analog of Trp of Xaa5 is a halogenated tryptophan suchas 5-fluoro-1-tryptophan or 6-fluoro-1-tryptophan. In some embodiments,the Gly between Xaa5 and Xaa6 is replaced with an N-methyl Gly (Sar).

Reference is made to the exemplary compstatin analogs of this class setforth below:

Cp30: (SEQ ID NO: 6) Sar-Ile-[Cys-Val-Trp(Me)-Gln-Asp-Trp-Sar-Ala-His-Arg-Cys]-mIle-NH₂ Cp40: (SEQ ID NO: 7)DTyr-Ile-[Cys-Val-Trp(Me)-Gln-Asp-Trp-Sar-Ala- His-Arg-Cys]-mIle-NH₂

As described in the examples herein, exemplary N- and C-terminallymodified analogs in accordance with the present invention were made bymodifying the analog Cp40.

TABLE 1 C-terminally modified analogs based on Cp40. Seq. Id. AnalogSequence* No. Cp40** DTyr-Ile-[Cys-Val-  7 Trp(Me)-Gln-Asn-Trp-Sar-Ala-His- Arg-Cys]-mIle-NH₂ Cp40-K DTyr-Ile-[Cys-Val-  8Trp(Me)-Gln-Asn- Trp-Sar-Ala-His- Arg-Cys]-mIle- Lys-NH₂ Cp40-KKDTyr-Ile-[Cys-Val-  9 Trp(Me)-Gln-Asn- Trp-Sar-Ala-His- Arg-Cys]-mIle-Lys-Lys-NH₂ Cp40-KKK DTyr-Ile-[Cys-Val- 10 Trp(Me)-Gln-Asn-Trp-Sar-Ala-His- Arg-Cys]-mIle- Lys-Lys-Lys-NH₂ *Brackets indicate aCys-Cys bond. **Cp40 is shown for comparison.PEGylation or the addition of Lys residues increased the solubility ofCp40 at physiological pH. Concurrently, the favorable C3 inhibitoryactivity of Cp40 was unaffected by the modifications, and, surprisingly,the binding affinity of the Lys derivatives towards C3 was even strongerthan that of the parental compound Cp40. In addition, the Cp40 variantsshowed similar or prolonged half-lives after subcutaneous, intravenous,intravitreal, and/or intramuscular administration into non-humanprimates, while the C3 concentrations were saturated for an extendedperiod of time when the Cp40 variants were used.

The most promising compounds of those assessed as described in theExamples, named mPEG(3k)-Cp40, Cp40-KK, and Cp40-KKK showed a drasticimprovement in their solubility (>200-fold) when compared to theparental peptide, Cp40. Most importantly, the novel analogs maintainedinhibitory activity and showed improved pharmacokinetic profiles whencompared to Cp40. In vivo studies in which non-human primates wereadministered sc with 2 mg/kg of the individual analogs indicated thatmPEG(3k)-Cp40 had a higher c_(max) (˜2-fold), longer C3 saturation time(˜34 h), slightly extended t_(1/2) by ˜15 h, increased AUC_(0-t)(˜2.7-fold) and decreased CL/F (˜3-fold) than did the Cp40 compound.Among the analogs with additional residues of Lys, Cp40-KKK appeared tobe the candidate with the most enhanced characteristics. This effect waseven more pronounced when Cp40-KKK was administered by multiple s.c.injections. In vivo pharmacokinetic studies showed that scadministration of Cp40-KKK was associated with higher c_(max) (˜2-fold),longer C3 saturation time (˜42 h), increased AUC_(0-t) (˜2.6-fold) anddecreased CL/F (˜3-fold) when compared to Cp40. Additionalpharmacokinetic studies of the individual analogues, showed that after asingle i.v. injection, Cp40-KKK exhibited an AUC₀₋₁₂₀ h value that wasover 3.5-fold greater than Cp40-KK and comparable to mPEG(1k)-Cp40 andmPEG(3k)-Cp40. In these i.v. PK studies, mPEG(3k)-Cp40 appeared to havethe most enhanced characteristics, showing an extended t_(1/2) by atleast ˜30 h, when compared to the other analogues. Moreover, in vivostudies in which non-human primates were administered intravitreally(i.v.t) with 0.5 mg of Cp40-KK, Cp40-KKK, mPEG(1k)-Cp40, ormPEG(3k)-Cp40 showed intravitreal residence times of at least 14 daysfor the PEGylated Cp40 analogs, with Cp40-KK and Cp40-KKK showingresidence times in excess of 73 days. Further still, Cp40-KKK exhibitedvitreous residence times at C3 saturating levels even after 90 days.

The overall improvement in solubility and pharmacokinetic profileassociated with these novel analogs is bolstered by their potential toreduce the frequency of drug administration and minimize localirritation at the injection site. In addition, the pharmacokineticstudies described herein indicate that the pharmacokinetic parametersfavoring long term systemic administration of these compounds, aremarkedly improved in comparison to Cp40.

Therefore, in particular embodiments, a compound is provided that isbased on SEQ ID No:7 in which at least one lysine amino acid iscovalently linked to the C-terminus. In some embodiments, at least twolysine amino acids are covalently linked to the C-terminus while inother embodiments, three or more lysine amino acids are covalently linedto the C-terminus. In preferred embodiments, a compound is provided thatis based on SEQ ID No:7 in which two or three lysine amino acids arecovalently linked to the C-terminus. In other embodiments, a compound isprovided that consists of SEQ ID NO:9 or SEQ ID NO:10. In still otherembodiments, the compound consists essentially of SEQ ID NO:9 or SEQ IDNO:10. For instance, in an embodiment, a compound is provided having anamino acid sequence represented by SEQ ID NO:10 (Cp40-KKK), wherein thecompound exhibits increased solubility, plasma residence time, vitreousresidence time, and/or C3-binding as compared to a compound having anamino acid sequence represented by SEQ ID NO:7 (Cp40).

The addition of small polymers, small PEGs and/or the C-terminaladdition of hydrophilic or charged residues can be applied to any otherclass of compstatin analog known in the art. These include, but are notlimited to, analogs described in WO2012/155107, WO2013/036778,WO2014/078731, WO2014/078734, WO2014/152931 and WO2017/062879.

The modified compstatin peptides of the present invention may beprepared by various synthetic methods of peptide synthesis viacondensation of one or more amino acid residues, in accordance withconventional peptide synthesis methods. For example, peptides aresynthesized according to standard solid-phase methodologies. Othermethods of synthesizing peptides or peptidomimetics, either by solidphase methodologies or in liquid phase, are well known to those skilledin the art. During the course of peptide synthesis, branched chain aminoand carboxyl groups may be protected/deprotected as needed, usingcommonly known protecting groups. Modification utilizing alternativeprotecting groups for peptides and peptide derivatives will be apparentto those of skill in the art.

Alternatively, certain peptides of the invention may be produced byexpression in a suitable prokaryotic or eukaryotic system. For example,a DNA construct may be inserted into a plasmid vector adapted forexpression in a bacterial cell (such as E. coli) or a yeast cell (suchas Saccharomyces cerevisiae), or into a baculovirus vector forexpression in an insect cell or a viral vector for expression in amammalian cell. Such vectors comprise the regulatory elements necessaryfor expression of the DNA in the host cell, positioned in such a manneras to permit expression of the DNA in the host cell. Such regulatoryelements required for expression include promoter sequences,transcription initiation sequences and, optionally, enhancer sequences.

The peptides can also be produced by expression of a nucleic acidmolecule in vitro or in vivo. A DNA construct encoding a concatemer ofthe peptides, the upper limit of the concatemer being dependent on theexpression system utilized, may be introduced into an in vivo expressionsystem. After the concatemer is produced, cleavage between theC-terminal Asn and the following N-terminal Gly is accomplished byexposure of the polypeptide to hydrazine.

The peptides produced by gene expression in a recombinant prokaryotic oreukaryotic system may be purified according to methods known in the art.A combination of gene expression and synthetic methods may also beutilized to produce compstatin analogs. For example, an analog can beproduced by gene expression and thereafter subjected to one or morepost-translational synthetic processes, e.g., to modify the N- orC-terminus or to cyclize the molecule.

Advantageously, peptides that incorporate unnatural amino acids, e.g.,methylated amino acids, may be produced by in vivo expression in asuitable prokaryotic or eukaryotic system. For example, methods such asthose described by Katragadda & Lambris (2006, Protein Expression andPurification 47: 289-295) to introduce unnatural Trp analogs intocompstatin via expression in E. coli auxotrophs may be utilized tointroduce N-methylated or other unnatural amino acids at selectedpositions of compstatin.

The structure of compstatin is known in the art, and the structures ofthe foregoing analogs are determined by similar means. Once a particulardesired conformation of a short peptide has been ascertained, methodsfor designing a peptide or peptidomimetic to fit that conformation arewell known in the art. Of particular relevance to the present invention,the design of peptide analogs may be further refined by considering thecontribution of various side chains of amino acid residues, as discussedabove (i.e., for the effect of functional groups or for stericconsiderations).

It will be appreciated by those of skill in the art that a peptide mimicmay serve equally well as a peptide for providing the specific backboneconformation and side chain functionalities required for binding to C3and inhibiting complement activation. Accordingly, it is contemplated asbeing within the scope of the present invention to produce C3-binding,complement-inhibiting compounds through the use of eithernaturally-occurring amino acids, amino acid derivatives, analogs ornon-amino acid molecules capable of being joined to form the appropriatebackbone conformation. A non-peptide analog, or an analog comprisingpeptide and non-peptide components, is sometimes referred to herein as a“peptidomimetic” or “isosteric mimetic,” to designate substitutions orderivations of the peptides of the invention, which possess the samebackbone conformational features and/or other functionalities, so as tobe sufficiently similar to the exemplified peptides to inhibitcomplement activation.

The use of peptidomimetics for the development of high-affinity peptideanalogs is well known in the art (see, e.g., Vagner et al., 2008, Curr.Opin. Chem. Biol. 12: 292-296; Robinson et al., 2008, Drug Disc. Today13: 944-951) Assuming rotational constraints similar to those of aminoacid residues within a peptide, analogs comprising non-amino acidmoieties may be analyzed, and their conformational motifs verified, byany variety of computational techniques that are well known in the art.

Uses and Therapeutic Administration of Compstatin Analogs

The complement inhibitory activity of compstatin analogs,peptidomimetics and conjugates may be tested by a variety of assaysknown in the art. In certain embodiments, the assays described in theExamples are utilized. A non-exhaustive list of other assays is setforth in U.S. Pat. No. 6,319,897, WO99/13899, WO2004/026328,WO2007/062249 and WO2010/127336, including, but not limited to, (1)peptide binding to C3 and C3 fragments; (2) various hemolytic assays;(3) measurement of C3 convertase-mediated cleavage of C3; and (4)measurement of Factor B cleavage by Factor D.

The peptides and peptidomimetics described herein are of practicalutility for any purpose for which compstatin itself is utilized, asknown in the art. Such uses include, but are not limited to: (1)inhibiting complement activation in the serum, and on cells, tissues ororgans of a patient (human or animal), which can facilitate treatment ofcertain diseases or conditions, including but not limited to,age-related macular degeneration, geographic atrophy, choroidalneovascularization, retinal neovascularization, ocular inflammation,hemodialysis-induced inflammation, glaucoma, uveitis, diabeticretinopathy rheumatoid arthritis, spinal cord injury, traumatic braininjury, cerebral ischemia/reperfusion injury (e.g. stroke), acutepolytrauma (hemorrhagic shock), Parkinson's disease, Alzheimer'sdisease, cancer, sepsis, paroxysmal nocturnal hemoglobinuria, hemolyticdisorders of autoimmune etiology (e.g. cold agglutinin disease andwAIHA), psoriasis and respiratory disorders such as asthma, chronicobstructive pulmonary disease (COPD), allergic inflammation, emphysema,bronchitis, bronchiectasis, cystic fibrosis, tuberculosis, pneumonia,respiratory distress syndrome (RDS—neonatal and adult), rhinitis andsinusitis, transplant-associated thrombotic microangiopathy, skininflammatory diseases, periodontitis, gingivitis, complement-associatedkidney diseases; (2) inhibiting complement activation that occurs duringcell or organ transplantation, or in the use of artificial organs orimplants (e.g., by time-restricted systemic administration before,during and/or after the procedure or by coating or otherwise treatingthe cells, organs, artificial organs or implants with a peptide of theinvention); (3) inhibiting complement activation that occurs duringextracorporeal shunting of physiological fluids (blood, urine) (e.g., bytime-restricted systemic administration before, during and/or after theprocedure or by coating the tubing through which the fluids are shuntedwith a peptide of the invention); and (4) in screening of small moleculelibraries to identify other inhibitors of compstatin activation (e.g.,liquid- or solid-phase high-throughput assays designed to measure theability of a test compound to compete with a compstatin analog forbinding with C3 or a C3 fragment).

To implement one or more of the utilities mentioned above, anotheraspect of the invention features pharmaceutical compositions comprisingthe compstatin analogs or conjugates described and exemplified herein.Such a pharmaceutical composition may consist of the active ingredientalone, in a form suitable for administration to a subject, or thepharmaceutical composition may comprise the active ingredient and one ormore pharmaceutically acceptable carriers, one or more additionalingredients, or some combination of these. The active ingredient may bepresent in the pharmaceutical composition in the form of aphysiologically acceptable ester or salt, such as in combination with aphysiologically acceptable cation or anion, as is well known in the art.

A particular compstatin analog of the invention may be selected for aparticular formulation on the basis of its solubility characteristics.As mentioned above, analogs that are highly soluble in water or bufferedsaline may be particularly suitable for systemic injection because theinjection volume can be minimized. By comparison, analogs with highwater solubility and lower solubility in buffered saline could produce amore long-lasting gel, suspension or precipitate for topical applicationor local injection, such as intraocular injection (includingintravitreal injection).

As such, in particular embodiments, the compstatin analogs based on Cp40are administered via subcutaneous, intravenous, intraocular (includingintravitreal), intramuscular injection, periodontal administration(including gingival administration or intrapapillary infiltrationinjection), or topical administration. In yet other embodiments, theCp40 analogs are delivered orally. In some embodiments, the Cp40-basedanalogs are administered as a single oral, subcutaneous, intravenous,intraocular (including intravitreal), intramuscular injection,periodontal administration, or topical administration. In otherembodiments, the Cp40-based analogs are administered via multiple oral,subcutaneous, intravenous, intraocular (including intravitreal),intramuscular injections, periodontal or topical administrations. In yetother embodiments, a Cp40-based analog is administered via an initialsubcutaneous, intravenous, or intramuscular loading dose combined withrepeated oral, intramuscular, subcutaneous or intravenous dosing for anextended period of time to allow for lower dosing of the analogs andless frequent dosing intervals as compared to administration methodspreviously described for known compstatin analogs. In still otherembodiments, maintenance dosing is accomplished via Cp40 analog deliveryby subcutaneous infusion pumps or ocular implants (see, e.g.,US2016/0060297, and U.S. Pat. No. 6,692,759).

The formulations of the pharmaceutical compositions may be prepared byany method known or hereafter developed in the art of pharmaceuticaltechnology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

The pharmaceutical compositions useful for practicing the invention maybe administered to deliver a dose of between 0.125 mg/kg and 50 mg/kgbody weight as a single bolus, or an intravitreal dose of between 1 gand about 10 mg, or in a repeated regimen, or a combination thereof asreadily determined by the skilled artisan. In certain embodiments, thedosage comprises at least 0.05 mg/kg, 0.1 mg/kg, or at least 0.2 mg/kg,or at least 0.3 mg/kg, or at least 0.4 mg/kg, or at least 0.5 mg/kg, orat least 0.6 mg/kg, or at least 0.7 mg/kg, or at least 0.8 mg/kg, or atleast 0.9 mg/kg, or at least 1 mg/kg, or at least 2 mg/kg, or at least 3mg/kg, or at least 4 mg/kg, or at least 5 mg/kg, or at least 6 mg/kg, orat least 7 mg/kg, or at least 8 mg/kg, or at least 9 mg/kg, or at least10 mg/kg, or at least 15 mg/kg, or at least 20 mg/kg, or at least 25mg/kg, or at least 30 mg/kg, or at least 35 mg/kg, or at least 40 mg/kg,or at least 45 mg/kg, or at least 50 mg/kg, on a daily basis or onanother suitable periodic regimen.

It has been discovered that administration of the Cp40-based analogsdisclosed herein (e.g., PEG(1K)-Cp40, PEG(3K)-Cp40, Cp40-KK, orCp40-KKK) have extended plasma residence times and extended intravitrealresidence times as compared to previously known compstatin analogs cantherefore be administered intravenously, intravitreally,intramuscularly, and/or subcutaneously at lower therapeuticallyeffective doses and less frequent dosing intervals. As one havingordinary skill in the art will appreciate, the specific route ofadministration may influence the dose required for therapeuticeffectiveness.

In one embodiment, the invention envisions intravenous or subcutaneousadministration of a Cp40-based analog, as described herein, at atherapeutically effective dose that is between about 0.125 mg/kg andabout 10 mg/kg, e.g., 0.125 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1mg/kg, 1.25 mg/kg, 1.5 mg/kg, 1.75 mg/kg, 2 mg/kg, 2.25 mg/kg, 2.5mg/kg, 2.75 mg/kg, 3 mg/kg, 3.25 mg/kg, 3.5 mg/kg, 3.75 mg/kg, 4 mg/kg,4.25 mg/kg, 4.5 mg/kg, 4.75 mg/kg, 5 mg/kg, 5.25 mg/kg, 5.5 mg/kg, 5.75mg/kg, 6 mg/kg, 6.25 mg/kg, 6.5 mg/kg, 6.75 mg/kg, 7 mg/kg, 7.25 mg/kg,7.5 mg/kg, 7.75 mg/kg, 8 mg/kg, 8.25 mg/kg, 8.5 mg/kg, 8.75 mg/kg, 9mg/kg, 9.25 mg/kg, 9.5 mg/kg, 9.75 mg/kg, or 10 mg/kg. In a preferredembodiment, the Cp40-based analog is administered via intravenous orsubcutaneous delivery (e.g., injection or infusion) at a therapeuticallyeffective dose that is between about 0.25 mg/kg and about 5 mg/kg. Inanother embodiment, the therapeutically effective dose is between about0.5 mg/kg and about 5 mg/kg. In yet another embodiment, thetherapeutically effective dose is between about 0.5 mg/kg and 4 mg/kg orbetween about 0.5 mg/kg and about 3 mg/kg. For instance, in oneparticular embodiment, the Cp40-based analog (e.g., PEG(1K)-Cp40,PEG(3K)-Cp40, Cp40-KK, or Cp40-KKK) is injected i.v. or s.c. to a humanat a dose of about 3 mg/kg/24 hours.

In another embodiment, the invention envisions intramuscularadministration of a Cp40-based analog, as described herein, at atherapeutically effective dose that is between about 0.25 mg/kg andabout 50 mg/kg, e.g., 0.25 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 7.5 mg/kg, 8 mg/kg, 8.5 mg/kg, 9mg/kg, 9.5 mg/kg, 10 mg/kg, 10.5 mg/kg, 11 mg/kg, 11.5 mg/kg, 12 mg/kg,12.5 mg/kg, 13 mg/kg, 13.5 mg/kg, 14 mg/kg, 14.5 mg/kg, 15 mg/kg, 15.5mg/kg, 16 mg/kg, 16.5 mg/kg, 17 mg/kg, 17.5 mg/kg, 18 mg/kg, 18.5 mg/kg,19 mg/kg, 19.5 mg/kg, 20 mg/kg, 20.5 mg/kg, 21 mg/kg, 21.5 mg/kg, 22mg/kg, 22.5 mg/kg, 23 mg/kg, 23.5 mg/kg, 24 mg/kg, 24.5 mg/kg, 25 mg/kg,26 mg/kg, 27 mg/kg, 28 mg/kg, 29 mg/kg, 30 mg/kg, 31 mg/kg, 32 mg/kg, 33mg/kg, 34 mg/kg, 35 mg/kg, 36 mg/kg, 37 mg/kg, 38 mg/kg, 39 mg/kg, 40mg/kg, 41 mg/kg, 42 mg/kg, 43 mg/kg, 44 mg/kg, 45 mg/kg, 46 mg/kg, 47mg/kg, 48 mg/kg, 49 mg/kg, or 50 mg/kg. In a preferred embodiment, theCp40-based analog is administered via intramuscular delivery (e.g.,injection) at a therapeutically effective dose that is between about0.25 mg/kg and about 35 mg/kg. In another embodiment, thetherapeutically effective dose is between about 0.25 mg/kg and 30 mg/kg.In yet another embodiment, the therapeutically effective dose is betweenabout 0.25 mg/kg and 10 mg/kg. In still other embodiments, thetherapeutically effective dose is between about 0.25 mg/kg and 5 mg/kg.For instance, in one particular embodiment, the Cp40-based analog (e.g.,PEG(1K)-Cp40, PEG(3K)-Cp40, Cp40-KK, or Cp40-KKK) is injected i.m. at adose of about 2.5 mg/kg

In yet another embodiment, the invention envisions intravitrealadministration of a Cp40-based analog, as described herein, at atherapeutically effective dose that is between about 1 g and about 10mg, e.g., 1 g, 1.25 g, 1.5 g, 1.75 g, 2 g, 2.25 g, 2.5 g, 2.75 g, 3 g,3.25 g, 3.5 g, 3.75 g, 4 g, 4.25 g, 4.5 g, 4.75 g, 5 g, 5.25 g, 5.5 g,5.75 g, 6 g, 6.25 g, 6.5 g, 6.75 g, 7 g, 7.25 g, 7.5 g, 7.75 g, 8 g,8.25 g, 8.5 g, 8.75 g, 9 g, 9.25 g, 9.5 g, 9.75 g, 10 g, 20 g, 30 g, 40g, 50 g, 60 g, 70 g, 80 g, 90 g, 100 g, 150 g, 200 g, 250 g, 300 g, 350g, 400 g, 450 g, 500 g, 550 g, 600 g, 650 g, 700 g, 750 g, 800 g, 850 g900 g, 950 g, 1 mg, 1.1 mg, 1.2 mg, 1.3 mg, 1.4 mg, 1.5 mg, 1.6 mg, 1.7mg, 1.8 mg, 1.9 mg, 2 mg, 2.1 mg, 2.2 mg, 2.3 mg, 2.4 mg, 2.5 mg, 2.6mg, 2.7 mg, 2.8 mg, 2.9 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, or 10 mg;preferably, the dose is between about 1 g and about 2,000 g, e.g., about1 g to about 2,000 g or about 100 g to about 1,500 g, or about 500 g toabout 1,200 g, or about 500 g to about 1,000 g. In some embodiments, thetherapeutically effective dose of Cp40-based analog is delivered viaintravitreal administration is at least about 0.02 mg, e.g., at leastabout 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg,0.09 mg, 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85, mg,0.9 mg, 0.95 mg, or 1 mg. For instance, in one particular embodiment,the Cp40-based analog (e.g., PEG(1K)-Cp40, PEG(3K)-Cp40, Cp40-KK, orCp40-KKK) is injected i.v.t. at a dose of about 1 mg.

In another embodiment, the invention envisions oral administration of aCp40-based analog, as described herein, at a therapeutically effectivedose that is between about 1 mg/kg and about 20 mg/kg, e.g., 1 mg/kg,1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 7.5 mg/kg, 8 mg/kg, 8.5mg/kg, 9 mg/kg, 9.5 mg/kg, 10 mg/kg, 10.5 mg/kg, 11 mg/kg, 11.5 mg/kg,12 mg/kg, 12.5 mg/kg, 13 mg/kg, 13.5 mg/kg, 14 mg/kg, 14.5 mg/kg, 15mg/kg, 15.5 mg/kg, 16 mg/kg, 16.5 mg/kg, 17 mg/kg, 17.5 mg/kg, 18 mg/kg,18.5 mg/kg, 19 mg/kg, 19.5 mg/kg, or 20 mg/kg. In a preferredembodiment, the Cp40-based analog is administered via oral delivery at atherapeutically effective dose that is between about 1 mg/kg and about10 mg/kg. For instance, in one particular embodiment, the Cp40-basedanalog (e.g., PEG(1K)-Cp40, PEG(3K)-Cp40, Cp40-KK, or Cp40-KKK) isdelivered orally to a human at a dose of about 1 and 5 mg/kg. In someembodiments, the oral dose described herein is administered once. Inother embodiments, it is administered daily.

In another embodiment, the invention envisions periodontaladministration, such as intrapapillary infiltration, of a Cp40-baseanalog, as described herein, at a therapeutically effective dose that isbetween about 1 g and about 1,000 g, e.g., 1 g, 5 g, 10 g, 15 g, 20 g,25 g, 30 g, 35 g, 40 g, 45 g, 50 g, 55 g, 60 g, 65 g, 70 g, 75 g, 80 g,85 g, 90 g, 95 g, 100 g, 110 g, 120 g, 130 g, 140 g, 150 g, 160 g, 170g, 180 g, 190 g, 200 g, 210 g, 220 g, 230 g, 240 g, 250 g, 260 g, 270 g,280 g, 290 g, 300 g, 310 g, 320 g, 330 g, 340 g, 350 g, 360 g, 370 g,380 g, 390 g, 400 g, 410 g, 420 g, 430 g, 440 g, 450 g, 460 g, 470 g,480 g, 490 g, 500 g, 550 g, 600 g, 650 g, 700 g, 750 g, 800 g, 850 g,900 g, 950 g, or 1,000 g. For example, the Cp40-based analog can beadministered periodontally to a human at a dose of between about 5 g andabout 500 g (e.g., delivered into the interdental papilla by injection).In a preferred embodiment, the Cp40-based analog is deliveredperiodontally to a human at a dose of between about 10 g/interdentalpapilla and about 200 g/interdental papilla or at a dose of betweenabout 20 g/interdental papilla and about 100 g/interdental papilla. Forinstance, in one particular embodiment, the Cp40-based analog (e.g.,PEG(1K)-Cp40, PEG(3K)-Cp40, Cp40-KK, or Cp40-KKK) is deliveredperiodontally to a human at a dose of about 25 g/interdental papilla orabout 50 g/interdental papilla.

In one embodiment, the invention envisions administration of a dose thatresults in a serum concentration of the Cp40-based analog between about0.01 μM and about 30 μM in an individual. In certain embodiments, thecombined dose and regimen will result in a serum concentration, or anaverage serum concentration over time, of the Cp40-based analog of atleast about 0.01 μM, or at least about 0.02 μM, or at least about 0.03μM, or at least about 0.04 μM, or at least about 0.05 μM, or at leastabout 0.06 μM, or at least about 0.07 μM, or at least about 0.08 μM, orat least about 0.09 μM, or at least about 0.1 μM, 0.11 μM, or at leastabout 0.12 μM, or at least about 0.13 μM, or at least about 0.14 μM, orat least about 0.15 μM, or at least about 0.16 μM, or at least about0.17 μM, or at least about 0.18 μM, or at least about 0.19 μM, or atleast about 0.2 μM, or at least about 0.3 μM, or at least about 0.4 μM,or at least about 0.5 μM, or at least about 0.6 μM, or at least about0.7 μM, or at least about 0.8 μM, or at least about 0.9 μM, or at leastabout 1 μM or at least about 1.5 μM, or at least about 2 μM, or at leastabout 2.5 μM, or at least about 3 μM, or at least about 3.5 μM, or atleast about 4 μM, or at least about 4.5 μM, or at least about 5 μM, orat least about 5.5 μM, or at least about 6 μM, or at least about 6.5 μM,or at least about 7 μM, or at least about 7.5 μM, or at least about 8μM, or at least about 8.5 μM, or at least about 9 μM, or at least about9.5 μM, or at least about 10 μM, or at least about 10.5 μM, or at leastabout 11 μM or at least about 11.5 μM, or at least about 12 μM, or atleast about 12.5 μM, or at least about 13 μM, or at least about 13.5 μM,or at least about 14 μM, or at least about 14.5 μM, or at least about 15μM, or at least about 15.5 μM, or at least about 16 μM, or at leastabout 16.5 μM, or at least about 17 μM, or at least about 17.5 μM, or atleast about 18 μM, or at least about 18.5 μM, or at least about 19 μM,or at least about 19.5 μM, or at least about 20 μM, or at least about20.5 μM, or at least about 21 μM or at least about 21.5 μM, or at leastabout 22 μM, or at least about 22.5 μM, or at least about 23 μM, or atleast about 23.5 μM, or at least about 24 μM, or at least about 24.5 μM,or at least about 25 μM, or at least about 25.5 μM, or at least about 26μM, or at least about 26.5 μM, or at least about 27 μM, or at leastabout 27.5 μM, or at least about 28 μM, or at least about 28.5 μM, or atleast about 29 μM, or at least about 29.5 μM, or at least about 30 μM.In certain embodiments, the combined dose and regimen will result in aserum concentration, or an average serum concentration over time, of theCp40-based analog of up to about 0.1 μM, or up to about 0.11 μM, or upto about 0.12 μM, or up to about 0.13 μM, or up to about 0.14 μM, or upto about 0.15 μM, or up to about 0.16 μM, or up to about 0.17 μM, or upto about 0.18 μM, or up to about 0.19 μM, or up to about 0.2 μM, or upto about 0.3 μM, or up to about 0.4 μM, or up to about 0.5 μM, or up toabout 0.6 μM, or up to about 0.7 μM, or up to about 0.8 μM, or up toabout 0.9 μM, or up to about 1 μM or up to about 1.5 μM, or up to about2 μM, or up to about 2.5 μM, or up to about 3 μM, or up to about 3.5 μM,or up to about 4 μM, or up to about 4.5 μM, or up to about 5 μM, or upto about 5.5 μM, or up to about 6 μM, or up to about 6.5 μM, or up toabout 7 μM, or up to about 7.5 μM, or up to about 8 μM, or up to about8.5 μM, or up to about 9 μM, or up to about 9.5 μM, or up to about 10μM, or up to about 10.5 μM or up to about 11 μM or up to about 11.5 μM,or up to about 12 μM, or up to about 12.5 μM, or up to about 13 μM, orup to about 13.5 μM, or up to about 14 μM, or up to about 14.5 μM, or upto about 15 μM, or up to about 15.5 μM, or up to about 16 μM, or up toabout 16.5 μM, or up to about 17 μM, or up to about 17.5 μM, or up toabout 18 μM, or up to about 18.5 μM, or up to about 19 μM, or up toabout 19.5 μM, or up to about 20 μM, or up to about 20.5 μM or up toabout 21 μM or up to about 21.5 μM, or up to about 22 μM, or up to about22.5 μM, or up to about 23 μM, or up to about 23.5 μM, or up to about 24μM, or up to about 24.5 μM, or up to about 25 μM, or up to about 25.5μM, or up to about 26 μM, or up to about 26.5 μM, or up to about 27 μM,or up to about 27.5 μM, or up to about 28 μM, or up to about 28.5 μM, orup to about 29 μM, or up to about 29.5 μM, or up to about 20 μM.

Suitable ranges include about 0.1 to about 30 μM, or about 1 to about 29μM, or about 2 to about 28 μM, or about 3 to about 27 μM, or about 4 toabout 26 μM, or about 5 to about 25 μM, or about 6 to about 24 μM, orabout 7 to about 23 μM, or about 8 to about 22 μM, or about 9 to about21 μM, or about 10 to about 20 μM, or about 11 to about 19 μM, or about12 to about 18 μM, or about 13 to about 17 μM, or about 1 to about 5 μM,or about 5 to about 10 μM, or about 10 to about 15 μM, or about 15 toabout 20 μM, or about 20 to about 25 μM, or about 25 to about 30 μM.While the precise dosage administered will vary depending upon anynumber of factors, including but not limited to, the type of patient andtype of disease state being treated, the age of the patient and theroute of administration, such dosage is readily determinable by theperson of skill in the art.

The pharmaceutical composition containing the Cp40-based analog can beadministered to a patient as frequently as several times daily, or itmay be administered less frequently, such as once a day, once a week,once every two weeks, once a month, or even less frequently, such asonce every several months or even once a year or less. The frequency ofthe dose will be readily apparent to the skilled artisan and will dependupon any number of factors, such as, but not limited to, the type andseverity of the disease being treated, the type and age of the patient,as described above. However, and as noted above, the Cp40-based analogsof the instant disclosure can be administered at less frequent intervalsas compared to previously known compstatin analogs.

For instance, in some embodiments, the intravenous, intramuscular,intraocular (including intravitreal), subcutaneous, periodontally(including gingival administration or intrapapillary infiltration) ortopical administration of a pharmaceutical composition containing theCp40-based analog is via a single injection. In other embodiments, theCp40-based analog is delivered orally. Additionally, and given theextended residence time of the presently described Cp40-based analogs(i.e., mPEGylated and/or Lys-containing Cp40-based analogs), theinvention envisions long term systemic administration of theseCp40-based analogs, wherein the Cp40-based analogs are delivered byoral, intravenous, intraocular (including intravitreal), subcutaneous,intramuscular, periodontally (including gingival administration orintrapapillary infiltration) or topical administration routes at theabove-described therapeutic doses via multiple deliveries (e.g., bymouth or injection) over time in order to provide a therapeuticallyeffective maintenance dose of the Cp40-based analogs depending on thetype and age of patient and the type and severity of disease treated.Thus, in some embodiments, a Cp40-based analog (e.g., PEG(1K)-Cp40,PEG(3K)-Cp40, Cp40-KK, or Cp40-KKK) is delivered intravenously,intraocularly (including intravitreally), subcutaneously,intramuscularly, periodontally (e.g., via intrapapillary infiltration),or topically, by multiple injections of a pharmaceutical compositioncomprising the analog administered once every about 12 hours to aboutonce every three months, e.g., once every 12 hours, once every 24 hours,once every 2 days, once every 3 days, once every 4 days, once every 5days, once every 6 days, once every 7 days, once every 8 days, onceevery 9 days, once every 10 days, once every 2 weeks, once every 3weeks, once every month, once every two months, once every three months.In other embodiments, the Cp40-based analog is delivered orally byingestion of a pharmaceutical composition comprising the analogadministered once every about 12 hours to about once every three months,e.g., once every 12 hours, once every 24 hours, once every 2 days, onceevery 3 days, once every 4 days, once every 5 days, once every 6 days,once every 7 days, once every 8 days, once every 9 days, once every 10days, once every 2 weeks, once every 3 weeks, once every month, onceevery two months, once every three months.

Also provided herein are delivery methods that include a combination ofadministration routes and doses. For instance, provided herein aremethods of systemic treatment of pharmaceutical compositions containingthe Cp40-based analogs of the instant disclosure (e.g., PEG(1K)-Cp40,PEG(3K)-Cp40, Cp40-KK, or Cp40-KKK) that includes an initial saturatingdose followed by multiple dosing at lower therapeutically effectivedoses and less frequent dosing intervals. This novel method of systemictreatment would provide for prolonged in vivo maintenance/control ofcomplement inhibition. To this end, in some embodiments, a first loadingdose of a Cp40-based analog is subcutaneously, intravenously, orintramuscularly administered at a higher therapeutically effective doseselected from the ranges described above, which is then followed byintramuscular or oral administration at regular dosing intervals. In onesuch embodiment, a pharmaceutical composition containing one of theCp40-based analogs discussed herein (e.g., Cp40-KKK) is injectedsubcutaneously or intravenously at an initial therapeutically effectivedose of at least about 0.5 to about 3 mg/kg and thereafter administeredorally or intramuscularly at a therapeutically effective maintenancedose of between about 0.25 mg/kg and about 50 mg/kg, wherein maintenancedose is delivered once every 2 days to 3 months, e.g., once every 2days, 3 days, 3 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days,19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days,27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days,35 days, 36 days, 37 days, 38 days, 39 days, 42 days, 45 days, 50 days,56 days, 60 days, 65 days, 70 days, 77 days, 80 days, 84 days, or 90days. For instance, in one particular embodiment, a pharmaceuticalcomposition containing Cp40-KKK is administered i.v. at a saturatingdose of about 0.5 mg/kg to about 3 mg/kg and then subsequentlyadministered i.m. weekly or every two weeks at a maintenance dose ofabout 0.5 to about 10 mg/kg.

As noted above, pharmaceutical compositions containing the Cp40-basedanalogs that are useful in the methods of the invention may beadministered systemically in oral, parenteral, ophthalmic or intraocular(including intravitreal), intravenous, subcutaneous, intramuscular(i.m.), periodontal (e.g., intrapapillary infiltration injection),suppository, aerosol, topical, transdermal or other similarformulations. Such pharmaceutical compositions may containpharmaceutically acceptable carriers and other ingredients known toenhance and facilitate drug administration. Other formulations, such asnanoparticles, liposomes, resealed erythrocytes, and immunologicallybased systems may also be used to administer a compstatin analogaccording to the methods of the invention.

Pharmaceutical compositions suitable for injectable use typicallyinclude sterile aqueous solutions (where water soluble) or dispersionsand sterile powders for the extemporaneous preparation of sterileinjectable solutions or dispersion. For intravenous administration,suitable carriers include physiological saline, bacteriostatic water,Cremophor EL™ (BASF, Parsippany, N.J.), phosphate buffered saline (PBS),or Ringer's solution.

Sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose, any bland fixed oil may be employedincluding synthetic mono- or di-glycerides. Fatty acids, such as oleicacid and its glyceride derivatives are useful in the preparation ofinjectables, as are natural pharmaceutically acceptable oils, such asolive oil or castor oil, especially in their polyoxyethylated versions.These oil solutions or suspensions may also contain a long-chain alcoholdiluent or dispersant, such as carboxymethyl cellulose or similardispersing agents that are commonly used in the formulation ofpharmaceutically acceptable dosage forms including emulsions andsuspensions. Other commonly used surfactants, such as Tweens, Spans andother emulsifying agents or bioavailability enhancers which are commonlyused in the manufacture of pharmaceutically acceptable solid, liquid, orother dosage forms may also be used for the purposes of formulation.

In general, the composition should be sterile, and should be fluid sothat easy syringability exists. Preferred pharmaceutical formulationsare stable under the conditions of manufacture and storage and may bepreserved against the contaminating action of microorganisms such asbacteria and fungi. In general, the relevant carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, sodium chloride in the composition. Prolongedabsorption of injectable compositions can be brought about by includingin the composition an agent which delays absorption, for example,aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Preferably solutions for injection are free ofendotoxin. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Formulations of a pharmaceutical composition suitable for oraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, in a variety of dosage forms,including but not limited to pills, tablets, granules, powders,capsules, dispersions, suspensions, solutions, emulsions,microemulsions, gels and films, to name a few. Such dosage formstypically include carriers, excipients, and or permeation enhancers tofacilitate formulation and delivery of the active ingredients.

The pharmaceutically acceptable carriers are selected from proteins,carbohydrates, lipids, organic and inorganic molecules, and combinationsthereof. The active ingredients can be combined with the carrier in anappropriate diluent to form a solution or a suspension. Such liquidformulations can be viscous or non-viscous depending on the amount andthe carrier used. The liquid formulations can be used directly or can befurther formulated into an appropriate capsule, gel capsule or solid bymethods know to those skilled in the art. Alternatively, solidformulations can be made by combining solid components. Such solidformulations can be used as a powder or formulated into granules,capsules, tablets or films any one of which can be made as a timerelease formulation.

Suitable proteins for use as carriers in oral dosage forms include milkproteins such as casein, sodium caseinate, whey, reduced lactose whey,whey protein concentrate, gelatin, soy protein (isolated), brown algaeprotein, red algae protein, baker's yeast extract and albumins. Suitablecarbohydrates include celluloses such as methylcellulose, sodiumcarboxymethylcellulose, carboxymethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, cellulose acetate and ethyl cellulose,starches such as cornstarch, potato starch, tapioca starch, wheatstarch, acid modified starch, pregelatinized starch and unmodifiedstarch, alginates such as ammonium alginate, sodium alginate, andcalcium alginate, glutens such as corn gluten and wheat gluten, gumssuch as acacia (gum Arabic), gum ghatti, guar gum, karaya gum (sterculiagum) and gum (tragacanth), insoluble glucose isomerase enzymepreparations, sugars such as corn sugar, invert sugar, corn syrup, highfructose corn syrup, and sodium gluconate. Suitable lipids includetocopherols such as a-tocopherol acetate, short-, medium- and long-chainfatty acids and esters thereof, fatty alcohols and ethers thereof, oilssuch as coconut oil (refined), soybean oil (hydrogenated) and rapeseedoil, aluminum palmitate, dilauryl thiodipropionate, enzyme-modifiedlecithin, calcium stearate, enzyme-modified fats, glycerylpalmitostereate, lecithin, mono- and diglycerides, glycerin and waxessuch as beeswax (yellow and white), candelilla wax and carnauba wax andvegetable oil. Suitable organic and inorganic substances include methyland vinyl pyrrolidones such as polyvinylpyrrolidone, methylsulfonylmethane, dimethylsulfoxide and related compounds, hydroxy andpolyhydroxy acids such as polylactic acid, among many others.

In some embodiments, oral dosage forms of the pharmaceuticalcompositions provided herein contain one or more permeation enhancersand/or lipid excipients to increase the bioavailability of thecompositions after oral administration such as those described in Maheret al. (2016, Adv. Drug Deliv. Rev. 106:277-319). Exemplary permeationenhancers suitable for use herein include C₁₂E₉, caprylocaproyl PEG 8glycerides, citric acid, dodecyl-β-D-maltopyranoside (DDM), glycerylmonocaprate, laurylocarnitine, n-tetradecyl β-D-maltopyranoside (TDM),N-trimethylated chitosan, palmitoylcarnitine, penetratin (D-penetratin),SNAC, sodium caprate (C₁₀), sodium caprylate (C₈), sodium cholate,sodium deoxycholate, sodium dodecyl sulphate, sodium taurocholate, andsucrose monolaurate. Exemplary lipid excipients suitable for use hereininclude polyoxylglycerides (e.g., polyoxyl stearate, polyethylene glycolmonostearate, caprylocaproyl polyoxyl-8 glycerides, caprylocaproylmacrogol-8 glycerides, lauraoyl polyoxylglycerides, stearoylpolyoxyglycerides, oleoyl polyoxyl-6 glycerides, linoleoyl polyoxyl-6glycerides, and lauroyl polyoxyl-6 glycerides), propylene glycol esters(e.g., propylene glycol monocaprylate type I, propylene glycolmonocaprylate type II, propylene glycol monolaurate type I, andpropylene glycol monolaurate type II), polyglycerol esters (e.g.,polyglyceryl-3 dioleate), glycerides (e.g., monoglycerides,diglycerides, glycerol monostearate 40-55 type I, medium chaintriglycerides, propylene glycol dicaprylate/dicaprate, propylene glycoldicaprylocaprate, glyceryl monolinoleate, and glyceryl monooleate type40), and hydroalcoholic solvents (e.g., diethylene glycol monoethylether).

In some embodiments, pharmaceutical compositions formulated for oraldelivery may include nanoparticles as a drug delivery system. Polymerssuitable for use in the coating of nano-carriers encapsulating the Cp-40analogs provided herein include, but are not limited to carbopol,chitosan, cholesteryl polymers, cyclodextrin, hydroxypropylmethylcellulose phthalate, poly(ethyl cyanoacrylate), polyethyleneglycol, polyacrylic acid, polylactide-co-glycolide, and polyallylamin(see, for example, Gupta et al., 2013, Drug Deliv. 20(6):237-246).

For topical applications, the pharmaceutical compositions providedherein may be formulated in a suitable ointment containing thepharmaceutically active component suspended or dissolved in one or morepharmaceutically acceptable carriers. Pharmaceutically acceptablecarriers for topical administration of the compstatin or compstatinanalogs disclosed herein include, but are not limited to, mineral oil,liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene,polyoxypropylene compound, emulsifying wax and water. Alternatively, thepharmaceutical compositions can be formulated in a suitable lotion orcream containing the active components suspended or dissolved in one ormore pharmaceutically acceptable carriers. Suitable carriers include,but are not limited to, mineral oil, sorbitan monostearate, polysorbate60, cetyl esters wax, cetearyl alcohol, 2 octyldodecanol, benzyl alcoholand water.

For local delivery to the eye, the pharmaceutical compositions providedherein may be appropriately formulated, for example (but not limitedto), in isotonic, pH adjusted sterile saline or water, either with orwithout a preservative such as benzylalkonium chloride. Alternatively,for ophthalmic uses, the pharmaceutical compositions may be formulatedin an ointment such as petrolatum or as eye drops.

Methods of local administration to the eye include, e.g., choroidalinjection, transscleral injection or placing a scleral patch, selectivearterial catheterization, eye drops or eye ointments, intraocularadministration including transretinal, subconjunctival bulbar,intravitreal injection, suprachoroidal injection, subtenon injection,scleral pocket and scleral cutdown injection, by osmotic pump, etc. TheCp40-analogs can also be alternatively administered intravascularly,such as intravenously (IV) or intraarterially. In choroidal injectionand scleral patching, the clinician or handler uses a local approach tothe eye after initiation of appropriate anesthesia, includingpainkillers and ophthalmoplegics. A needle containing the pharmaceuticalcomposition is directed into the subject's choroid or sclera andinserted under sterile conditions. When the needle is properlypositioned, the Cp40 analog is injected into either or both of thechoroid or sclera. When using either of these methods, the clinician orhandler can choose a sustained release or longer acting formulation.Thus, the procedure can be repeated only every several months or severalyears, depending on the subject's tolerance of the treatment andresponse.

Intraocular administration of drugs is well known in the art. See, e.g.,U.S. Pat. Nos. 5,632,984 and 5,770,589 and U.S. Pub. No. 2016/0060297A1. U.S. Pat. No. 6,378,526 provides methods for intrascleral injectionof a therapeutic or diagnostic material at a location overlying theretina, which provide a minimally invasive technique for delivering theagent to the posterior segment of the eye.

In certain embodiments, a pharmaceutical composition containing a Cp40analog of the present invention is delivered to the vicinity of the eye,e.g., in close proximity to the posterior segment of the eye. The“vicinity of the eye” refers to locations within the orbit, which is thecavity within the skull in which the eye and its appendages aresituated. Typically the compositions would be delivered close to theirintended target within the eye, e.g., close to (within severalmillimeters of) the portion of the sclera that overlies the posteriorsegment of the eye, or immediately adjacent to the exterior surface ofthe sclera. In a preferred embodiment, the pharmaceutical compositionsof the present invention are delivered into the vitreous cavity of theeye (i.e., intravitreally).

A number of polymeric delivery vehicles for providing controlled releasehave been used in an ocular context and can be used to administer thepharmaceutical compositions of the invention. Various polymers, e.g.,biocompatible polymers, which may be biodegradable, can be used. Forexample, U.S. Pat. No. 6,692,759 describes methods for making animplantable device for providing controlled release of therapeuticagents in the eye. Other useful polymers and delivery systems for ocularadministration of a therapeutic agent have been described. The activeagent may be released as the polymer degrades. Polymers that have beenused for drug delivery include, but are not limited to,poly(lactic-co-glycolic acid), polyanhydrides, ethylene vinyl acetate,polyglycolic acid, chitosan, polyorthoesters, polyethers, polylacticacid, and poly (beta amino esters). Peptides, proteins such as collagenand albumin, and dendrimers (e.g., PAMAM dendrimers) have also beenused. Any of these can be used in various embodiments of the invention.

Poly(ortho esters) have been introduced into the eye and demonstratedfavorable properties for sustained release ocular drug delivery (seeEinmahl, S., 2002, Invest. Ophthalmol. Vis. Sci. 43(5)). Polylactideparticles have been used to target an agent to the retina and RPEfollowing intravitreal injection of a suspension of such particles(Bourges et al., 2003, Invest. Ophthalmol. Vis. Sci. 44(8)). Amacroscopic implantable device suitable for introduction into theposterior or anterior segment of the eye is referred to herein as anocular implant (see Jaffe, G., 2000, Invest. Ophthalmol. Hs. Sci.,41(11)). Therefore, provided herein is an ocular implant comprising aCp40 analog, e.g., in a therapeutically effective amount to deliver theCp40 analog to the individual with a disease or condition treatable bycomplement inhibition. Such devices may be macroscopic implantscomprising the Cp40 analog or may be comprised of a plurality ofnanoparticles or microparticles impregnated with or encapsulating theagent. In one embodiment, the ocular implant is any ocular implant knownin the art. Exemplary implants and methods for manufacture thereof aredescribed, e.g., in US 2009/0220572 A1. Other implants known in the artcan also be used.

Other embodiments include gel-forming compositions comprising a solublecollagen that are useful for the delivery of compstatin or compstatinanalogs to the posterior segment of the eye. The collagen is initiallysoluble and forms a solution that has a low viscosity but is capable ofrapid formation of a gel under appropriate conditions, e.g., conditionsencountered upon administration to a mammalian subject. The inventiontherefore provides a system for delivery of the pharmaceutically activeagents to the posterior segment of the eye. The system is designed tolocalize such molecules in sufficient concentration to provide sustaineddelivery while at the same time allowing the macromolecule to bereleased in sufficient amounts. In addition, the collagen gel mayprotect the compstatin or compstatin analogs from degradation, e.g., byendogenous proteases.

The composition forms a gel following introduction into the body, e.g.,upon contact with a physiological fluid. The composition can also form agel upon contact with a fluid such as phosphate buffered saline, orother fluid containing appropriate ions. Thus the composition can beinjected at an appropriate location, e.g., in close proximity to theposterior segment of the eye, where it forms a gel. Alternately, apreshaped gel implant can be made, e.g., by introducing the solutioninto a mold or cavity of the desired shape and allowing gel formation tooccur in the presence of a suitable concentration of a salt. The saltcan be added either prior to or following the introduction of thesolution into the mold or cavity. The mold or cavity can be, e.g., anystructure that contains a hollow space or concave depression into whicha solution can be introduced. In another embodiment, a film or membraneis formed from the collagen solution containing a therapeutic agent.

Release of the agent from the gel can occur by any mechanism, e.g., bydiffusion of the agent out of the gel, as a result of breakdown of thegel, or both. One aspect of the invention is the selection of suitableconcentrations of soluble collagen and collagen solids that result in agel that retains the agent within the gel so as to provide sustaineddelivery for a desired period of time while also permitting release ofthe agent from the gel in sufficient concentration to be effective atits site of action in the posterior segment of the eye.

In accordance with certain embodiments of the invention, a solutioncontaining the soluble collagen and compstatin or a compstatin analog isprepared by combining the soluble collagen and the compstatin orcompstatin analog in solution using any suitable method, e.g., by addingthe compstatin or compstatin analog to a solution containing solublecollagen. The composition is delivered locally to an appropriatelocation in or near the eye of a mammalian subject, typically to an areaoutside of and in close proximity to the posterior segment of the eye.The solution rapidly forms a gel at or close to of the site ofadministration. The compstatin or compstatin analog is entrapped withinthe gel and then diffuses out of the gel or is released as the geldegrades over time, thereby providing a continuous supply of thecompstatin or compstatin analog to tissues and structures that areeither in direct physical contact with the gel or located nearby ordelivering into the blood stream. In certain embodiments the solution isadministered behind the sclera of the eye, as discussed further below.Delivery can be accomplished by injection (e.g., using a 30 gauge needleor the like), by catheter, etc., as further described below.

A variety of different collagen preparations can be used in the presentinvention provided that the collagen is initially soluble and is capableof rapidly forming a gel under appropriate conditions. Suitable collagenpreparations, and methods for their manufacture, are described, e.g., inU.S. Pat. Nos. 5,492,135; 5,861,486; 6,197,934; 6,204,365; and WO00/47130, but the invention is not limited to such preparations ormethods. These collagens are prepared in soluble form and rapidly form agel upon exposure to physiological fluids or other fluids havingsuitable concentration of ions. In accordance with the presentinvention, injecting or otherwise introducing the collagen solution tothe eye or near the eye results in gel formation, presumably induced bycontact with physiological fluids. However it is noted that theinvention is in no way limited by the mechanism by which gel formationoccurs. In addition, as noted above, the gel can be formed in vitro andthem implanted at an appropriate location, e.g., in close proximity tothe posterior segment of the eye.

One suitable method of preparing a soluble collagen solution involvesextracting collagen from a natural source, acid solubilizing thecollagen, and dialyzing the solubilized collagen against a solutioncontaining a chelating agent, e.g., a metal chelating agent such asethylenediamine tetraacetic acid, disodium salt dihydrate (EDTA), whileraising the pH. One or more dialysis steps against a solution such asdeionized water lacking the chelating agent may also be performed.Unlike standard collagen solutions that undergo spontaneousfibrillogenesis at neutral pH and room temperature, collagen solutionsfor use in the present invention remain in solution during storage forextended periods of time and rapidly undergo gel formation when exposedto physiological fluids. While not wishing to be bound by any theory,the chelating agent may alter the concentration of one or more cationsand thereby prevent fibrillogenesis that would otherwise occur as the pHis raised. The chelating agent may have other desirable effects on thecollagen solution, and in certain embodiments of the invention thecollagen solution comprises a chelating agent, e.g., EDTA. The chelatingagent may remain in the collagen solution following dialysis or may beadded to the collagen solution. The concentration of the chelating agentmay range, for example, between about 0.02M and about 0.05M, e.g.,between about 0.025M and about 0.035M. Other chelating agents may alsobe used including, but not limited to, those described in U.S. Pat. No.5,861,486.

In certain embodiments the collagen solution has a concentration ofsoluble collagen ranging between 1 mg/ml and 100 mg/ml, e.g., between 10mg/ml and 70 mg/ml, between 20 mg/ml and 50 mg/ml, e.g., 30 mg/ml, etc.In certain embodiments of the invention the pH of the collagen solutionis between 6.0 and 8.0, e.g., between 6.5 and 7.5, e.g., 7.0.

In certain embodiments of the invention the collagen composition furthercomprises a fibrillar component comprising fibrillar collagen solids.For example, certain collagen compositions contain between 0.5 mg/ml and30 mg/ml fibrillar collagen solids, or between 1 mg/ml and 20 mg/mlfibrillar collagen solids, e.g., 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6mg/ml, 8 mg/ml, 10 mg/ml, etc. In terms of percent fibrillar collagensolids on a weight/volume basis, certain collagen compositions containbetween 0.05 and 3% fibrillar collagen solids or between 0.1 and 2%fibrillar collagen solids, e.g., 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.8%, 1%,1.2%, etc. Any suitable fibrillar component can be used in the collagencompositions of the invention. Fibrillar collagen solids can be preparedusing a variety of methods. For example, fibrillar collagen may bereconstituted collagen prepared from animal sources such as bovine hide(Frontiers in Matrix Biology, Vol. 10, pp. 1-58, in Methods ofConnective Tissue Research, Eds. Robert, Moczar, and Moczar, S. Karger,Basel, 1985). Fibrillar collagen may be prepared from human or animalsources as described in U.S. Pat. Nos. 4,969,912 and 5,322,802. Thefibrillar collagen solids are suspended in solution at a concentrationtypically ranging from about 10-100 mg/ml. The collagen suspensioncontaining fibrillar collagen solids is combined with, e.g., added to, asoluble collagen composition either prior to or following addition ofthe therapeutic agent to a solution comprising soluble collagen.

In some embodiments of the invention the soluble collagen preparationcomprises a chemical cross-linking agent. The agent may crosslinkcollagen molecules and/or fibrils to one another and/or may crosslink atherapeutic agent such as compstatin or an analog thereof to a collagenmolecule or fibril. Typical cross-linking agents crosslink collagenamine groups to one another or to amine, carboxyl, phenol, sulfonyl, orcarbohydrate groups of therapeutic agents. Suitable cross-linking agentsinclude, but are not limited to, those described in WO 00/47130. Withoutwishing to be bound by any theory, cross-linking may stabilize thecollagen gel (e.g., decrease its rate of breakdown) and/or decrease therate of release of the therapeutic agent from the gel.

Without wishing to be bound by any theory, the presence of fibrillarcollagen solids may have any of a variety of advantageous effects. Byway of non-limiting example, the fibrillar collagen solids may increasethe in vivo stability of the collagen gel, e.g., they may decrease therate of breakdown of the gel. The fibrillar collagen solids may increasethe stability of a therapeutic agent contained in the gel and/ordecrease or modulate the rate at which the agent is released from thegel by diffusion and/or breakdown of the gel.

The collagen preparations preferably form a gel within 5 minutes (300seconds) following contact with physiological fluids. More preferablythe collagen preparations form a gel within 90 seconds, 2 minutes (120seconds) or within 3 minutes (180 seconds) following contact withphysiological fluids. Preparations that form a gel within shorter timeperiods, e.g., within 5-90 seconds, or longer time periods, e.g., 3-5minutes, can also be used.

Any of collagen types I-XXVIII, or mixtures thereof, can be used in thepresent invention. The collagen can be purified from natural sources(e.g., human tissue or animal tissue such as bovine, rabbit, etc.) asdescribed in the above-referenced patents and publications.Alternatively, the collagen can be manufactured using recombinant DNAtechniques, in which case the sequence can be of human or animal origin.See, e.g., U.S. Pat. Nos. 5,593,854 and 5,667,839. Methods for theproduction of proteins, e.g., a polypeptide of interest such as acollagen chain, using recombinant DNA technology are well known in theart. Suitable methods include those described above. The term “collagen”includes collagen fragments. Thus in certain embodiments the solublecollagen comprises or consists of a collagen fragment or combination offragments. In certain embodiments a complete collagen polypeptide chainis used.

While collagen preparations such as those described above areparticularly preferred in certain embodiments of the invention, avariety of other gel-forming materials could also be used in agel-forming composition of the invention. In certain embodiments the gelis a hydrogel, by which is meant a gel that contains a substantialamount of water. Preferably the material and the gel that it forms arebiocompatible. In certain embodiments the material and the gel that itforms are biodegradable. A variety of modified or derivatized collagensare also of use in various embodiments of the invention. See, e.g., U.S.Pat. No. 5,201,764. For example, collagen can be acylated with one ormore acylating agents such as glutaric anhydride, succinic anhydride,and maleic anhydride and at least one other acylating agent selectedfrom the group consisting of methacrylic anhydride, beta-styrenesulfonyl chloride, ethylene-maleic anhydride copolymer, styrene-maleicanhydride copolymer or poly(vinyl) sulfonic acid.

Other gel-forming materials include, but are not limited to, hyaluronicacid and modified forms thereof, polysaccharides such as alginate andmodified forms thereof, self-assembling peptides, etc. See, e.g., U.S.Pat. No. 6,129,761 for further description of alginate and modifiedforms thereof, hyaluronic acid and modified forms thereof, andadditional examples of soluble gel-forming materials that are of use invarious embodiments of the present invention. As described therein,other polymeric hydrogel precursors include polyethyleneoxide-polypropylene glycol block copolymers such as Pluronics™ orTetronics™ which are crosslinked by hydrogen bonding and/or by atemperature change, as described in Steinleitner et al., Obstetrics &Gynecology, 77:48-52 (1991); and Steinleitner et al., Fertility andSterility, 57:305-308 (1992). Other materials which may be utilizedinclude proteins such as fibrin or gelatin. Polymer mixtures also may beutilized. For example, a mixture of polyethylene oxide and polyacrylicacid which gels by hydrogen bonding upon mixing may be utilized.

Covalently crosslinkable hydrogel precursors also are useful. Forexample, a water soluble polyamine, such as chitosan, can becross-linked with a water soluble diisothiocyanate, such as polyethyleneglycol diisothiocyanate. The isothiocyanates will react with the aminesto form a chemically crosslinked gel. Aldehyde reactions with amines,e.g., with polyethylene glycol dialdehyde also may be utilized. Ahydroxylated water soluble polymer also may be utilized.

Alternatively, polymers may be utilized which include substituents whichare crosslinked by a radical reaction upon contact with a radicalinitiator. For example, polymers including ethylenically unsaturatedgroups which can be photochemically crosslinked may be utilized, asdisclosed in WO 93/17669, the disclosure of which is incorporated hereinby reference. In this embodiment, water soluble macromers that includeat least one water soluble region, a biodegradable region, and at leasttwo free radical-polymerizable regions, are provided. The macromers arepolymerized by exposure of the polymerizable regions to free radicalsgenerated, for example, by photosensitive chemicals and or light.Examples of these macromers are PEG-oligolactyl-acrylates, wherein theacrylate groups are polymerized using radical initiating systems, suchas an eosin dye, or by brief exposure to ultraviolet or visible light.Additionally, water soluble polymers which include cinnamoyl groupswhich may be photochemically crosslinked may be utilized, as disclosedin Matsuda et al., ASAID Trans., 38:154-157 (1992).

In general, the polymers are at least partially soluble in aqueoussolutions, such as water, buffered salt solutions, or aqueous alcoholsolutions. Methods for the synthesis of the other polymers describedabove are known to those skilled in the art. See, for example ConciseEncyclopedia of Polymer Science and Polymeric Amines and Ammonium Salts,E. Goethals, editor (Pergamen Press, Elmsford, N.Y. 1980). Manypolymers, such as poly(acrylic acid), are commercially available.Naturally occurring and synthetic polymers may be modified usingchemical reactions available in the art and described, for example, inMarch, “Advanced Organic Chemistry,” 4th Edition, 1992,Wiley-Interscience Publication, New York.

Water soluble polymers with charged side groups may be crosslinked byreacting the polymer with an aqueous solution containing ions of theopposite charge, either cations if the polymer has acidic side groups oranions if the polymer has basic side groups. Examples of cations forcrosslinking of the polymers with acidic side groups to form a hydrogelare monovalent cations such as sodium, and multivalent cations such ascopper, calcium, aluminum, magnesium, strontium, barium, and tin, anddi-, tri- or tetra-functional organic cations such as alkylammoniumsalts. Aqueous solutions of the salts of these cations are added to thepolymers to form soft, highly swollen hydrogels and membranes. Thehigher the concentration of cation, or the higher the valence, thegreater the degree of cross-linking of the polymer. Additionally, thepolymers may be crosslinked enzymatically, e.g., fibrin with thrombin.In some embodiments a self-assembling peptide, such as those describedin U.S. Pat. No. 6,800,481 is used. These peptides self-assemble to forma hydrogel structure upon contact with monovalent cations, e.g., such asthose present in extracellular fluid.

In embodiments of the invention in which the gel is formed bycross-linking polymer chains to one another, the composition can includean appropriate cross-linking agent, which is selected according to theparticular polymer. Alternately, the cross-linking agent can beadministered after administration of the composition containing thegel-forming material, at substantially the same location. Any of thesegels can be formed in vitro, e.g., as described above for gelscomprising soluble collagen, and implanted at an appropriate location inor in the vicinity of the eye.

In certain embodiments, the implants described herein comprise betweenabout 100 g and about 50 mg of a Cp40 analog, e.g., between about 100 gand about 40 mg between about 100 g and about 30 mg between about 100 gand about 20 mg between about 100 g and about 10 mg between about 100 gand about 9 mg e.g., between about 100 g and about 8 mg, e.g., betweenabout 100 g and about 7 mg, e.g., between about 100 g and about 6 mg,e.g., between about 100 g and about 5 mg, e.g., between about 100 g andabout 4 mg, e.g., between about 100 g and about 3 mg, e.g., betweenabout 100 g and about 2 mg, e.g., between about 100 g and about 1 mg,e.g., between about 100 g and about 500 g.

Methods for making microparticles and nanoparticles are known in theart. Generally, a microparticle will have a diameter of 500 microns orless, e.g., between 50 and 500 microns, between 20 and 50 microns,between 1 and 20 microns, between 1 and 10 microns, and a nanoparticlewill have a diameter of less than 1 micron. Preferably the device isimplanted into the space occupied by the vitreous humor. The ocularimplant may comprise a polymeric matrix. The invention also providesperiocular implants, which are macroscopic implantable devices suitablefor introduction in the vicinity of the eye, e.g., in close proximity tothe eye. In certain embodiments the periocular implant is made ofsimilar materials to those described above.

In other embodiments, cells that express a Cp40 analog can be implantedinto the eye. U.S. Pat. No. 6,436,427 provides a method for deliveringbiologically active molecules to the eye by implanting biocompatiblecapsules containing a cellular source of the biologically activemolecule.

In some embodiments, controlled release forms may be prepared to achievea sustained, or location-specific liberation of the compstatin analog inthe digestive tract in order to improve absorption and prevent certainforms of metabolism. For example, acid-resistant coatings of tablet oracid-resistant capsule materials may be used to prevent a release ofcompstatin analogs in the stomach and protect the compound frommetabolism by gastric enzymes. Suitable materials and coatings toachieve controlled release after passage of the stomach are primarilycomposed of fatty acids, waxes, shellac, plastics and plant fibers andinclude, but are not limited to, methyl acrylate-methacrylic acidcopolymers, cellulose acetate succinate, hydroxy propyl methyl cellulosephthalate, hydroxy propyl methyl cellulose acetate succinate(hypromellose acetate succinate), polyvinyl acetate phthalate, sodiumalginate or stearic acid. Sustained release in the gastrointestinaltract can for example be achieved by embedding compstatin analogs in amatrix of insoluble substances such as various acrylics, chitin andothers. Methods to prepare such formulations are known to those skilledin the art.

Compstatin may be formulated into suppositories or clysters for rectal,vaginal or urethral administration. For this purpose, compstatin analogscan be dissolved or suspended in a greasy base carrier such as cocoabutter that is solid or semi-solid at room temperature but melts at bodytemperature or in a water-soluble solid base such as polyethylene glycolor glycerin (made from glycerol and gelatin). Other excipients may beadded to improve the formulation, and suppositories will be shaped in aform that facilitates administration. In other embodiments, liquidsuppositories consisting of compstatin analogs dissolved or suspended ina liquid carrier suitable for rectal delivery to be applied with a smallsyringe may be used.

For the treatment of chronic or acute lung conditions in whichcomplement activation is implicated, a preferred route of administrationof a pharmaceutical composition is pulmonary administration.Accordingly, a pharmaceutical composition of the invention may beprepared, packaged, or sold in a formulation suitable for pulmonaryadministration via the buccal cavity. Such a formulation may comprisedry particles which comprise the active ingredient and which have adiameter in the range from about 0.5 to about 7 nanometers, andpreferably from about 1 to about 6 nanometers. Such compositions areconveniently in the form of dry powders for administration using adevice comprising a dry powder reservoir to which a stream of propellantmay be directed to disperse the powder or using a self-propellingsolvent/powder-dispensing container such as a device comprising theactive ingredient dissolved or suspended in a low-boiling propellant ina sealed container. Preferably, such powders comprise particles whereinat least 98% of the particles by weight have a diameter greater than 0.5nanometers and at least 95% of the particles by number have a diameterless than 7 nanometers. More preferably, at least 95% of the particlesby weight have a diameter greater than 1 nanometer and at least 90% ofthe particles by number have a diameter less than 6 nanometers. Drypowder compositions preferably include a solid fine powder diluent suchas sugar and are conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50 to 99.9% (w/w) of the composition, and theactive ingredient may constitute 0.1 to 20% (w/w) of the composition.The propellant may further comprise additional ingredients such as aliquid non-ionic or solid anionic surfactant or a solid diluent(preferably having a particle size of the same order as particlescomprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery may also provide the active ingredient in the form of dropletsof a solution or suspension. Such formulations may be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, including replacement pulmonary surfactant, or a preservativesuch as methylhydroxybenzoate. The droplets provided by this route ofadministration preferably have an average diameter in the range fromabout 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary deliveryare also useful for intranasal delivery of a pharmaceutical compositionof the invention.

Another formulation suitable for intranasal administration is a coarsepowder comprising the active ingredient and having an average particlefrom about 0.2 to 500 micrometers. Such a formulation is administered inthe manner in which snuff is taken i.e. by rapid inhalation through thenasal passage from a container of the powder held close to the nares.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient, and may further comprise one or more of theadditional ingredients described herein.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, intravenous,subcutaneous, intraperitoneal, intramuscular, intraarticular,intravitreal, intrasternal injection, and kidney dialytic infusiontechniques.

Formulations of a pharmaceutical composition suitable for parenteraladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e. powder or granular) form for reconstitution with asuitable vehicle (e.g. sterile pyrogen-free water) prior to parenteraladministration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution can be formulated according to the knownart, and may comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations may beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active ingredient inmicrocrystalline form, in a liposomal preparation, in microbubbles forultrasound-released delivery or as a component of a biodegradablepolymer systems. Compositions for sustained release or implantation maycomprise pharmaceutically acceptable polymeric or hydrophobic materialssuch as an emulsion, an ion exchange resin, a sparingly soluble polymer,or a sparingly soluble salt.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agentsincluding replacement pulmonary surfactants; dispersing agents; inertdiluents; granulating and disintegrating agents; binding agents;lubricating agents; sweetening agents; flavoring agents; coloringagents; preservatives; physiologically degradable compositions such asgelatin; aqueous vehicles and solvents; oily vehicles and solvents;suspending agents; dispersing or wetting agents; emulsifying agents,demulcents; buffers; salts; thickening agents; fillers; emulsifyingagents; antioxidants; antibiotics; antifungal agents; stabilizingagents; and pharmaceutically acceptable polymeric or hydrophobicmaterials. Other “additional ingredients” which may be included in thepharmaceutical compositions of the invention are known in the art anddescribed, for example in Genaro, ed., 1985, Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa.

Methods:

Another aspect of the invention features methods of regulatingcomplement activation. In general, the methods comprise contacting amedium in which regulation of complement activation is desired with acompstatin analog of the present invention, wherein the contactingresults in regulation of complement activation in the medium. The mediumcan be any medium in which regulation of complement activation isdesired. In certain embodiments, the medium includes cells or tissues ofan organism, including (1) cultured cells or tissues, (2) cells ortissues within the body of a subject or patient, and (3) cells ortissues that have been removed from the body of one subject and will bereplaced into the body of the same patient (e.g., extracorporealshunting of blood or autologous transplantation) or transferred toanother patient. In connection with the latter embodiment, the mediummay further comprise a biomaterial, such as tubing, filters or membranesthat contact the cells or tissues during extracorporeal shunting.Alternatively, the medium may comprise biomaterials that are implantedinto a subject.

In certain embodiments, the methods of regulating complement activationapply to living patients or subjects and comprise part or all of amethod of treating the patient for a pathological condition associatedwith complement activation, particularly AP-mediated complementactivation, which can amplify complement effector responses andexacerbate inflammatory damage in tissues and cells, irrespective of thetriggering mechanism of complement activation. Many such pathologicalconditions are known in the art (see, e.g., Holers, 2008, supra) andinclude, but are not limited to, atypical hemolytic uremic syndrome(aHUS); dense deposit disease (DDD); C3 glomerulonephritis (C3GN); C3glomerulopathies; other complement-mediated nephropathies and glomerularinflammatory diseases; age-related macular degeneration (AMD); any eyedisorder characterized by macular degeneration, choroidalneovascularization (CNV); retinal Neovascularization (RNV),proliferative vitreoretinopathy, glaucoma, uveitis, ocular inflammation,or any combination of these; paroxysmal nocturnal hemoglobinuria (PNH);cold agglutinin disease (CAD); warm antibody autoimmune hemolyticanemias (wAIHAs); sickle cell disease; transplant-associated thromboticmicroangiopathies; rheumatoid arthritis (RA), systemic lupuserythematosus (SLE); several autoimmune and autoinflammatory kidneydiseases; autoimmune myocarditis; multiple sclerosis; traumatic brainand spinal cord injury; cerebral, intestinal and renalischemia-reperfusion (IR) injury; spontaneous and recurrent pregnancyloss; antiphospholipid syndrome (APS); Parkinson's disease; Alzheimer'sdisease; other neurodegenerative inflammatory conditions underpinned byaberrant synaptic remodeling, excessive microglial activity andcognitive decline; asthma; anti-nuclear cytoplasmic antigen-associatedpauci-immune vasculitis (Wegener's syndrome); non-lupus autoimmune skindiseases such as pemphigus, bullous pemphigoid, and epidermolysisbullosa; post-traumatic shock, cancer; periodontitis; gingivitis; andatherosclerosis. In particular embodiments, the pathological conditionhas been associated with mutations and polymorphisms in the geneencoding FH and/or CD46, including but not limited to: AMD, aHUS andmembrano-proliferative glomerulonephritis type II (MPGN-II, alsoreferred to as dense deposit disease (DDD)). In other embodiments, thecompstatin analogs of the present invention are suitable for use as asubstitute for Eculizumab in treatment of diseases for which thoseagents are currently prescribed, or for which they are being developedin pre-clinical and clinical studies. Those diseases include, but arenot limited to, aHUS, PNH, C3G (DDD/C3GN), CAD and AMD.

The treatment methods typically comprise (1) identifying a subject witha disease or condition treatable by regulation of complement activationas described hereinabove, (2) measuring a parameter of the disease orcondition treatable by regulation of complement activation usingart-standard techniques well within the purview of the skilled artisan(e.g., biopsy, histology, MRI, bone-scan, X-Ray, pain tolerance,posture, and the like), (3) administering to the subject an effectiveamount of a compstatin analog of the invention using a treatment regimenand duration appropriate for the condition being treated, and (4)measuring the parameter of the disease or condition as an indicationthat the disease or condition has been ameliorated or has been treated.Delivery of the compstatin or compstatin analog may be performed by anysuitable route of administration known in the art, including orally,nasal (e.g., via nasal spray), intraocular (including intravitreal),rectal, intravenous injection/infusion, subcutaneous injection/infusion,intramuscular, periodontal (e.g., gingival administration orintrapapillary infiltration), topical and the like. Development ofappropriate dosages and treatment regimens will vary depending upon anynumber of factors, including but not limited to, the type of patient andtype of disease state being treated, the age of the patient and theroute of administration. The skilled artisan is familiar with the designof dosage regimens that take such variables into account. For instance,it will be apparent to the skilled artisan that oral administration of acompstatin analog of the invention will require a higher initial dosage,due to the lesser bioavailability from that route as compared with,e.g., intravenous injection. Likewise, intramuscular administration of acompstatin analog of the invention would require a higher dose thandelivery of the same analog via intravenous or intravitreal injection.Suitable therapeutically effective doses are described in more detailelsewhere herein.

In one embodiment, a method for treating an individual, such as a humanpatient or non-human primate, with a disease or condition treatable byregulation of complement activation is provided that includes the stepsof first identifying an individual with the disease or conditiontreatable by regulation of complement activation and then administeringto the individual a therapeutically effective amount of a Cp40-basedanalog of the invention, wherein the route of administration isintravenous or subcutaneous, and wherein the therapeutically effectiveamount of the Cp40-based analog is between about 0.125 mg/kg to about 10mg/kg; preferably, the amount is between about 0.25 mg/kg and about 5mg/kg, or between about 0.5 mg/kg and about 5 mg/kg, or between about0.5 mg/kg and about 4 mg/kg, or between about 0.5 mg/kg and about 3mg/kg, or about 3 mg/kg. In another embodiment, the route ofadministration is intramuscular, and the therapeutically effectiveamount of the Cp40-based analog is between about 0.25 mg/kg to about 50mg/kg; preferably, the amount is between about 0.25 mg/kg and about 35mg/kg, or between about 0.25 mg/kg and about 30 mg/kg, or between about0.25 mg/kg and about 10 mg/kg, or between about 0.25 mg/kg and about 5mg/kg, or about 2.5 mg/kg. In some aspects, the route of administrationis orally, and the therapeutically effective amount of the Cp40-basedanalog is between about 1 mg/kg to about 20 mg/kg; preferably, theamount is between about 1 mg/kg and about 10 mg/kg or between about 1mg/kg and about 5 mg/kg. In another embodiment, the route ofadministration is intravitreal, and the therapeutically effective amountof the Cp40-based analog is between about 1 g to about 10 mg;preferably, the amount is between about 1 g and about 2,000 g or about 1mg. Other suitable therapeutically effective doses and routes ofadministration are described in more detail elsewhere herein. In theseembodiments, the method also includes one or more measuring steps thatincludes measuring at least one parameter of the disease or conditiontreatable by regulation of complement activation using art-standardtechniques well within the purview of the skilled artisan (e.g., biopsy,histology, MRI, bone-scan, X-Ray, pain tolerance, posture, and thelike), whereby measuring the parameter of the disease or condition maybe used as an indication that the disease or condition has been treated,it being understood that the measuring step can be performed before,during, and/or after administering the Cp40-based analog.

In another embodiment, a method for treating an individual with adisease or condition treatable by regulation of complement activation isprovided that includes administering to the individual an initialtherapeutically effective amount of a Cp40-based analog of theinvention, wherein the route of administration is intravenous orsubcutaneous, and wherein the initial therapeutically effective amountof the Cp40-based analog is at least about 0.125 mg/kg to about 10mg/kg; preferably, the amount is between about 0.5 mg/kg to about 3mg/kg. This treatment is then followed by administering to theindividual a maintenance dose of a Cp40-based analog of the invention,wherein the route of administration is intramuscular, and wherein themaintenance dose of the Cp40-based analog is at between about 0.25 mg/kgand about 50 mg/kg; preferably, it is between about 0.25 mg/kg and about10 mg/kg. Alternatively, the maintenance dose is administered orally,and the maintenance does of the Cp40-based analog is between about 1mg/kg and about 20 mg/kg; preferably, it is between about 1 mg/kg andabout 10 mg/kg. Other routes of administering the maintenance dose arealso described elsewhere herein. The maintenance dose is thenadministered via multiple deliveries given once every 2-3 days to aboutonce every 1 month; preferably, once every 2 weeks. Alternatively, theinitial therapeutically effective dose is at least about 2 mg/kgadministered via an intramuscular route. In these embodiments, themethod also includes one or more measuring steps that includes measuringat least one parameter of the disease or condition treatable byregulation of complement activation using art-standard techniques wellwithin the purview of the skilled artisan (e.g., biopsy, histology, MRI,bone-scan, X-Ray, pain tolerance, posture, and the like), wherebymeasuring the parameter of the disease or condition may be used as anindication that the disease or condition has been treated, it beingunderstood that the measuring step can be performed before, during,and/or after administering the Cp40-based analog.

Also provided herein are novel methods of generating antibodies againstcompstatin analogs. The method typically includes immunization of asuitable animal for generating antibodies, such as a rat, mouse, monkey,rabbit, goat, pig, or sheep. In a preferred embodiment, a rabbit isimmunized with a compstatin analog; preferably, the compstatin analog isCp40 or a Cp40-based analog (e.g., Cp40-KK or Cp40-KKK). In suchaspects, the compstatin analog is conjugated with a suitable carrierprotein, such as keyhole limpet hemocyanin (KLH), prior to immunization.In other embodiments, the compstatin analog is conjugated to the carrierprotein in a mixture further comprising an adjuvant; preferably, it is astrong adjuvant. Typical adjuvants includes inorganic compounds (e.g.,alum, aluminum hydroxide, aluminum phosphate, calcium phosphatehydroxide), mineral oil, detergents, plant saponins, cytokines (e.g.,IL-1, IL-2, IL-12), block copolymers, and combinations thereof. Forinstance, in one particular embodiment, a rabbit is immunized with Cp40or a Cp40-based analog conjugated to KLH in the presence of a strongadjuvant. The immunization dose ranges from about 50 g to about 500 g;preferably, the dose is about 100 g. The immunized animal is thenadministered the analog-KLH-adjuvant composition via injection every 2days to every 4 weeks; preferably, the animal is injected weekly. Theinjections are continued for at least 2 weeks, e.g., 2 weeks, 3 weeks, 4weeks, 5 weeks, or more. In a preferred embodiment, the immunized animalis given 4 weeks injections of the analog-KLH-adjuvant composition for 5weeks. The injections comprise dose ranges from 10 g to about 100 g;preferably, the dose is about 50 g. The Cp40-specific antibodies arethen purified from the animal serum by any suitable protein purificationtechnique known in the art, e.g., affinity chromatography. The Cp40 andCp40-Lysine antibodies produced by the method described herein arehighly specific for the Cp40 or Cp40-Lysine peptide as described infurther detail in Example 7 below. Thus, these novel antibodies cannotonly specifically detect the different compstatin analogs, but canfurther discriminate between the unmodified and lysine-modified versionsthereof. In a preferred embodiment, the novel antibodies are monoclonalantibodies.

Also provided herein are novel and sensitive methods for the detectionof the Cp40-based analogs described herein, in biological fluidsincluding those from which scanty samples of minimal volume can only beretrieved, such as the vitreous. Such methods require only very smallquantities of biological fluid. These methods employ SPR analysisperformed on vitreous samples or plasma samples containing theCp40-based analogs of interest. In one embodiment, a method of detectinga Cp40-based analog in a biological sample is provided that includes thesteps of (1) providing a biological sample that contains Cp40-basedanalog molecules, where at least a portion of these analogs are bound toC3/C3b/C3c; (2) providing a CM5 sensor chip to which a plurality of Cp40or Cp40 analog molecules are covalently attached; (3) heat-inactivatingthe biological sample to release the compstatin analogs from the targetmolecules (i.e., C3/C3b/C3c); (4) mixing the heat-inactivated samplewith a pre-determined amount of C3 of human plasma as a source of C3;(5) contacting the mixture of the heat-inactivated sample with a fixedamount of C3 or of human plasma to the CM5 sensor chip, whereby the Cp40analog molecules released from the target-bound Cp40 complexes in thebiological sample, will compete with the Cp40 molecules/analogsimmobilized on the CM5 sensor chip for binding to C3 or plasma-derivedC3; and (6) detecting the free C3 by binding to Cp40 or its analogsimmobilized on the CM5 sensor chip. The SPR signal is directlyproportional to the amount of C3 bound to the immobilized Cp40 moleculesor its analogs. An SPR signal reduction therefore corresponds to thereduction of binding of free C3 to the immobilized Cp40 and serves as ameasure of the amount of free Cp40 or Cp40 analogs present in theheat-inactivated biological sample. Quantification of the unknown Cp40analog amount in the sample is performed according to a standard curveof known peptide concentrations. In a preferred embodiment, the Cp40molecules that are covalently attached to the CM5 sensor chip are thesame Cp40-based analog molecules of interest in the biological sample.For instance, in a particular embodiment, Cp40-KKK is covalentlyattached to the CM5 sensor chip and the biological sample (e.g., avitreous sample) is first heat-inactivated, as described above, andsubsequently mixed with a calibrated source of C3 and flowed over thechip to allow the competitive detection of peptide released fromC3/C3b/C3c-bound Cp40-KKK complexes in the sample.

The following examples are provided to describe the invention in greaterdetail. They are intended to illustrate, no to limit, the invention.

Example 1. Design of Analogs Based on Cp40

Cp40(DTyr-Ile-[Cys-Val-Trp(Me)-Gln-Asn-Trp-Sar-Ala-His-Arg-Cys]-mIle-NH₂)(SEQ ID NO:7), Cp40-Lys-NH₂ (Cp40-K; SEQ ID NO: 8), Cp40-Lys-Lys-NH₂(Cp40-KK; SEQ ID NO: 9), and Cp40-Lys-Lys-Lys-NH₂ (Cp40-KKK; SEQ ID NO:10) were synthesized in house or by GL Biochem (Shanghai, China) usingFmoc-solid phase peptide synthesis and purified using reversed-phasehigh-performance liquid chromatography (RP-HPLC) based on techniquesknown in the art (see, e.g., Qu, H. et al., 2011, Mol Immunol48:481-489; Qu, H. et al., 2013, Immunobiology 218:496-505). Asillustrated in FIG. 2, the purity of the compounds was verified byRP-HPLC and matrix-assisted laser desorption ionization-time-of-flightmass spectrometry (MALDI-TOF MS) on a Micromass® MALDI micro MX™ (WatersCorporation, Milford, Mass.). Depending on the amount of compound, anXBridge BEH C18 column (5-μm particle size, 150-mm length, WatersCorporation, Milford, Mass.) with either a diameter of 4.6, 10, or 19 mmwas used for RP-HPLC, and elution was achieved with a gradient of 5-70%acetonitrile in 0.1% aqueous TFA solution within 30 min at a flow rateof 2, 4.5, or 16 ml/min, respectively.

The synthesis of mPEG(3k)-, mPEG(2k)-, mPEG(1k)-, and mPEG(1056)-Cp40was carried out based on PEGylation procedures known in the art (U.S.Pat. No. 8,962,553; Risitano et al., 2014, Blood 123:2094-2101). Inbrief, 1 eq of Cp40 (5 mg/ml) was dissolved in acetonitrile/water (1:1),and 2 eq of the respective activated mPEG ester were added. The pH ofthe reaction mixture was adjusted to 8 using N-methylmorpholine. Afterstirring for 0.5-1 h at room temperature, the reaction mixture wasquenched upon addition of 0.1% aqueous TFA (pH 2). All peptides werepurified by RP-HPLC as described above and characterized by MALDI-TOF MS(see FIG. 2).

For the preparation of mPEG(528)-Cp40, linear Cp40 was synthesized onresin as described previously (see, e.g., Qu, H. et al., 2011, MolImmunol 48:481-489; Qu, H. et al., 2013, Immunobiology 218:496-505).After Fmoc-deprotection of the final N-terminal amino acidFmoc-DTyr(tBu)-OH by use of 20% piperidine in DMF, 3 eq of mPEG(528)-NHSester in DMF were added to the dried beads. The pH was adjusted to 8.0using N-methylmorpholine, and agitation was allowed to proceed for 2 hon a rotator. Reaction completion was confirmed by Kaiser test. Thebeads were subsequently washed with DMF and DCM and dried under vacuum.The peptide was cleaved from resin as described by Qu et al. (supra).The lyophilized crude linear deprotected peptide (1 eq) was dissolved in80% aqueous methanol, and 20 mM iodine (13 eq) in methanol was slowlyadded with vigorous stirring. After 30 min of stirring at roomtemperature, the cyclization reaction was quenched by the addition of 20mM aqueous ascorbic acid. Methanol was removed under reduced pressureand the crude peptide was purified by RP-HPLC as described above. Thepeptide was then characterized by MALDI-TOF MS (see FIG. 2).

All peptides were initially obtained as a TFA salt and further convertedinto an acetate salt on the HPLC column using 25 mM aqueous ammoniumacetate as described in EP 2163558 A3. The mass of the final compoundswas confirmed by MALDI-TOF MS. Peptides used for in vivo experimentswere tested for the presence of endotoxin (<0.03 EU/ml). Cp40, Cp40-K,Cp40-KK, and Cp40-KKK were synthesized by GL Biochem (Shanghai, China)using Fmoc-solid phase peptide synthesis and purified usingreversed-phase high-performance liquid chromatography (RP-HPLC) based ona procedure described previously by Qu et al. The purity of thecompounds was verified by RP-HPLC and MALDI-TOF MS (FIG. 2) on aMicromass® MALDI micro MX™ (Waters Corporation, Milford, Mass.).Depending on the amount of compound, an XBridge BEH C18 column (5-μmparticle size, 150-mm length, Waters Corporation, Milford, Mass.) witheither a diameter of 4.6, 10, or 19 mm was used for RP-HPLC, and elutionwas achieved with a gradient of 5-70% acetonitrile in 0.1% aqueous TFAsolution within 30 min at a flow rate of 2, 4.5, or 16 ml/min,respectively.

Example 2. Solubility of Cp40-Based Analogs

To test the solubility of the Cp40-based analogs designed in Example 1,for each analog, 5-10 mg of the peptide was weighed in a LoBindEppendorf tube and mixed with 20 μl of PBS (pH 7.4). The mixture wasvortexed and centrifuged for 2 min at 16,873×g. Unless otherwise noted,if the respective peptide was not dissolved, PBS was added in portionsof 10 μl, followed by vortexing and centrifugation, until allprecipitate disappeared. The pH of the peptide solutions was determinedon indicator paper (Whatman™ 2614-991, Type CF, wide-range pH teststrips with colorimetric chart, pH range 4.5-10, size 6×80 mm). Theconcentrations of the final solutions were determined based on theabsorbance measured on a NanoDrop™ 2000c spectrophotometer (ThermoScientific, Wilmington, Del.) at 280 nm using the equation c=A/ε·b(c=concentration, A=absorbance, ε=extinction coefficient, b=pathlength).

While Cp40 is highly solubility in water, it is less soluble atphysiological pH (0.8 mg/ml in PBS at pH 7.4) (Qu et al., 2013,Immunobiology 218:496-505). Therefore, to create a Cp40 with improvedsolubility without significantly decreasing the inhibitory activity orbinding affinity for C3, Cp40 was modified at either its N-terminalregion or its C-terminal region as described in Example 1.

Previous reports indicated that Cp40 modified with a 40 kDa PEG chain atits C-terminus was associated with a drop in inhibitory activity,whereas the Cp40 modified with a 40 kDa PEG chain at its N-terminusexhibited an extended residence time in plasma after in vivoadministration in NHP, but had greater than 100-fold lower bindingaffinity as compared to unmodified Cp40 (Risitano et al., 2014, Blood123:2094-2101; see FIGS. 2 and 3). In the present study, the N-terminusof the Cp40 was coupled to polydisperse PEG chains of 1, 2, or 3 kDa viaamide coupling. The resulting analogs were termed mPEG(3k)-Cp40,mPEG(2k)-Cp40, and mPEG(1k)-Cp40 (FIG. 1). PEGylation greatly increasedthe solubility of the resulting Cp40-based analogs, with mPEG(3k)-Cp40showing the highest solubility in PBS, 270 mg/ml (pH=7.0, Table 2).

Compounds PEGylated with polydisperse polymers have a less-definedstructure making them more difficult to characterize (Veronese, 2001,Biomaterials 22:405-417). To create a better defined-structure that canbe better characterized, PEGylation of Cp40 using monodisperse polymerswere carried out using the activated NHS esters of mPEG(1056) andmPEG(528). Whereas PEGylation using polydisperse PEGs and mPEG(1056) wasperformed with pre-synthesized Cp40, PEGylation of the monodispersecompound mPEG(528)-Cp40 was carried out on-resin, eliminating oneadditional step of HPLC purification. The solubility properties of theresulting monodisperse analog mPEG(1056)-Cp40 were found to be verysimilar to those of its polydisperse counterpart. In contrast,attachment of a smaller PEG chain (528 Da) resulted in a significantdrop in the solubility, from about 140 to 2.3 mg/ml in PBS (Table 2).

In addition to PEGylation, incorporation of hydrophilic/charged residueswas used as an alternative approach to increase the solubility of Cp40.To this end, one, two, or three lysine residues were attached to theC-terminus of Cp40 during the peptide synthesis. Notably, compoundsolubility increased with an increasing number of Lys residues, i.e.,the solubility of Cp40-K (37 mg/ml) was much less than that of Cp40-KKand Cp40-KKK (>245 mg/ml) (Table 2). Notably, whereas a pH of 7-7.5 wasmeasured in solutions of 277 mg/ml Cp40-KK in PBS, the presence of thethird Lys residue led to a higher pH in the respective Cp40-KKKsolutions. A pH in the 7 range was observed only with a Cp40-KKKconcentration of 7 mg/ml (Table 2).

TABLE 2 Solubility and pH of the Cp40-based analogs in PBS. Analog Conc.(mg/ml) pH Cp40    1.08 7.5 mPEG(3k)-Cp40 >270 7 mPEG(2k)-Cp40   172 7.5mPEG(1k)-Cp40   157 7-7.5 mPEG(1056)-Cp40   137 7-7.5 mPEG(528)-Cp40   2.3 7-7.5 Cp40-K    7.1 7.5 Cp40-KK >272 7-7.5 Cp40-KKK >245 8.5Cp40-KKK    7.0 7.5* Cp40-KKK <100** 9.5 *pH adjusted upon addition ofPBS. **after additional lyophilization from H₂O.

Example 3. Complement Inhibitory Potency and Target Affinity ofCp40-Based Analogs

To determine whether the modified peptides retained Cp40's inhibitoryactivity and target affinity, inhibition of the classical pathway ofcomplement activity was evaluated in an in vitro enzyme-linkedimmunosorbent assay (ELISA) (see, for example, Mallik, 2005, Journal ofMedicinal Chemistry 48:274-286). In brief, antigen-antibodycomplex-mediated complement activation in normal human plasma in thepresence or absence of Cp40 and its analogs was detected based on C3bdeposition. For this purpose, microtiter wells (NUNC) were coated with50 μl of 1% ovalbumin in PBS (pH 7.4) at ambient temperature for 2 h.The wells were blocked with 200 μl of 1% BSA in PBS for 1 h, then coatedwith 50 μl of 1:1000 α-ovalbumin polyclonal antibody in PBS for 1 h.Between each step, the plate was washed thrice with 200 μl of 0.05%Tween 20 in PBS (PBS-T); 30 μl of VBS (Veronal buffer 1×; 5 mM veronal,pH 7.4, containing 150 mM NaCl, 0.5 mM CaCl₂) and 0.5 mM MgCl₂) wereplaced in all but the first well of each row of the 96-well plate, and 5μM peptide solutions in VBS were prepared. Peptide concentrations weredetermined on a Nanodrop™ 2000c spectrophotometer from Thermo Scientificat 280 nm using an extinction coefficient of 12,615·M⁻¹ cm⁻¹ for allpeptides. Human plasma diluted 1:40 in VBS was incubated for 15 min withpeptide of 0.005 to 2.5 μM. After washing with PBS-T, 50 μl/well of1:1000 goat α-human C3 HRP conjugated antibody in 1% BSA in PBS wasadded to the wells and incubated for 1 h at room temperature. Complementfixation indicating activation was detected by the addition of HRPsubstrate (0.05% ABTS and 0.1% of 30% aqueous H₂O₂ in 0.1 M sodiumcitrate, pH 4.2) and read at 405 nm. The absorbance data obtained at 405nm were translated into % inhibition, considering 100% to be equal tocomplement activation in the absence of peptide. The percent inhibitionwas plotted against the logarithm of concentrations, and the resultingdata set was fitted to the equation “log(inhibitor) vs. normalizedresponse” using GraphPad Prism 5 (La Jolla, Calif.). IC₅₀ values wereobtained from the fitted parameters of the mean of at least threeindependent experiments. Cp40 was always used as the internal control.

The binding affinities and kinetic profiles of the Cp40-based analogswith C3b were assessed by SPR using a Biacore 3000 instrument (GEHealthcare, Piscataway Township, N.J.) based on previously describedprotocols (see, e.g., Qu et al., 2011, Mol Immunol 48:481-489; Qu etal., 2013, Immunobiology 218:496-505; Magotti et al., 2009, J MolRecognit 22:495-505; Huang et al., 2014, Chem Med Chem 9:2223-2226). Allexperiments were carried out at 25° C. using 0.01 M HEPES, pH 7.4, with0.15 M NaCl, 3 mM EDTA, and 0.005% surfactant P20 (HBS-EP) as therunning buffer. Purified human C3b (Complement Technology, Inc., Tyler,Tex.) was coated onto a CM5 sensor chip (GE Healthcare, Uppsala, Sweden)at densities of 11,000-20,000 resonance units (RUs) via amide coupling,as adapted from the immobilization procedure provided with the AmineCoupling Kit from GE Healthcare. A non-coated flow cell was used as areference surface. A series of five samples of increasing concentrationsof each Cp40 (2.5, 5, 10, 20, 40 nM) analog were successively injectedfor 2 min each at a flow rate of 30 μl/min, with a final dissociationstep of 80 min. Cp40 was included in every SPR experiment as an internalcontrol. Each peptide was screened in at least three independentexperiments. All sensorgrams were processed using Scrubber software(BioLogic Software, Campbell, Australia). The resulting data wereglobally fitted to a 1:1 Langmuir binding model in BIAevaluationsoftware (GE Healthcare) to obtain the equilibrium dissociation constant(K_(D)) from the equation K_(D)=k_(d)/k_(a).

As shown in Table 3 and FIG. 3, the inhibitory activity of the modifiedCp40 peptide was not significantly influenced by PEGylation at theN-terminus or the addition of Lys residues at the C-terminus. Incontrast, the binding affinity of the individual peptides to C3b wasaffected by the Cp40 modifications (Table 3). Whereas the addition ofthe shortest PEG chain to the N-terminus of Cp40 (mPEG(528)-Cp40)resulted in a 5-fold drop in the binding affinity toward C3b, increasingthe length of the PEG chain further decreased the affinity ofmPEG(3k)-Cp40 (K_(D) 7.9 nM) as compared to that of Cp40 (K_(D) 0.5 nM)(Table 3 and FIGS. 4 and 5). On the other hand, attachment of Lysresidues had a positive effect on the binding of the resulting analogsto C3b. Whereas the addition of a single Lys residue did not affect thebinding significantly, addition of two or three Lys residues resulted ina 1.25- and 2.5-fold increase in the binding affinity to C3b,respectively (Table 3 and FIGS. 4 and 5). In general, the variation inthe association rate ((0.7−4)×10⁶M⁻¹ s⁻¹) of the various analogs washigher than that of the dissociation rate ((1.2−2.8)×10⁻³ s⁻¹).

TABLE 3 Inhibitory activity and C3b binding affinity of Cp40-basedanalogs.* IC₅₀ k_(a) k_(d) K_(D) Analog (nM) (10⁶M⁻¹s⁻¹) (10⁻³s⁻¹) (nM)Cp40 56.8 ± 3.57 ± 1.84 ± 0.53 ± 3.0 0.70 0.40 0.14 mPEG(3k)-Cp40 83.0 ±0.45 ± 2.40 ± 7.91 ± 7.0 0.27 0.91 2.02 mPEG(2k)-Cp40 83.8 ± 0.54 ± 2.39± 4.47 ± 4.5 0.13 0.46 0.61 mPEG(1k)-Cp40 52.7 ± 0.76 ± 2.18 ± 2.98 ±2.9 0.15 0.14 0.54 mPEG(1056)-Cp40 52.0 ± 0.72 ± 2.80 ± 3.93 ± 2.7 0.250.95 0.39 mPEG(528)-Cp40 83.5 ± 1.00 ± 2.45 ± 2.58 ± 8.1 0.27 0.28 0.51Cp40-K 95.6 ± 1.98 ± 1.85 ± 0.92 ± 9.2 0.20 0.50 0.20 Cp40-KK 61.8 ±3.94 ± 1.52 ± 0.44 ± 4.2 1.26 0.25 0.17 Cp40-KKK 81.7 ± 3.83 ± 1.24 ±0.21 ± 5.3 2.58 0.42 0.09 k_(a), association constant k_(d),dissociation constant K_(D), equilibrium dissociation constant *Allvalues were calculated from the man of at least three independentexperiments.

Example 4. Pharmacokinetic Analysis of Cp40-Based Analogs in Plasma ofNon-Human Primates after Subcutaneous Administration

Methods.

Cp40, mPEG(3k)-Cp40, Cp40-KK, and Cp40-KKK were tested in vivo in NHPsto assess the influence of PEGylation and Lys conjugation on theirpharmacokinetic profiles. The studies were performed at the SimianConservation Breeding and Research Center (SICONBREC), Inc. (Makati,Philippines). Each peptide analog (Cp40, Cp40-KK, Cp40-KKK,mPEG(3k)-Cp40) was tested in two individual 6- to 7-year-old, healthymale cynomolgus monkeys (Macaca fascicularis), with a body weight ofabout 4 kg. Each peptide was administered at 2 mg/kg in a singlesubcutaneous injection: 8 mg of net Cp40 were dissolved in 2 ml ofsterile saline (Cp40, Cp40-KK), 0.5 ml of sterile saline(mPEG(3k)-Cp40), or 0.25 ml of 100 mM phosphate buffer (Cp40-KKK) andinjected subcutaneously using a 3/10-mL insulin safety syringe with29GX1/2″ needle. Blood samples were collected before (0 h) and atvarious time points after the sample administration (t=5 min, 30 min, 1,2, 4, 6, 12, 24, 48, 72, 96, 120 h) into EDTA-vacutainer bloodcollecting tubes to prevent coagulation and complement activation. Allblood samples were centrifuged at ˜800×g for 10 min, and the resultingplasma samples were immediately frozen and shipped to the University ofPennsylvania for further analysis. All NHP studies were performed inaccordance with animal welfare laws and regulations.

To analyze the plasma samples and determine the plasma half-life,preparation of standard solutions and plasma samples was firstperformed. Calibration curves were prepared together with the plasmasamples being analyzed: Stock solutions of the respective peptide (Cp40,Cp40-KK, Cp40-KKK, or mPEG(3k)-Cp40) were spiked into untreated NHPplasma at final concentrations of 1, 2, 4, 8, and 16 μM. Theconcentrations of the stock solutions were determined using a Nanodrop™2000c spectrophotometer from Thermo Scientific at 280 nm. Prior tofurther analysis, all plasma samples were treated with methanol forprotein precipitation as follows: 500 of the NHP plasma to be analyzedwas mixed in a 0.5 ml LoBind Eppendorf tube with 150 μl methanolcontaining 0.5 μg/ml isotope-labeled Cp40(DTyr-Ile-[Cys-Val-Trp(Me)-Gln-Asn-Trp-Sar-Ala-His-[13C6;15N4]Arg-Cys]-mIle-NH₂,Bachem, Torrance, Calif.), which served as internal standard (IS). Themixture was vortexed for ˜8 min, allowed to sit at room temperature for10 min and centrifuged for 20 min at 16,873×g. The supernatant was mixed1:1 with a solution of 20% methanol in 10 mM aqueous ammonium formate(pH 3) in an injection vial (TruView LCMS Certified Clear Glass 12×32 mmScrew Neck Total Recovery Vial, Waters Corporation, Milford, Mass.). FormPEG(3k)-Cp40 samples, 3% aqueous MeCN was used instead of the ammoniumformate solution.

For liquid chromatography and mass spectrometry, plasma samples wereprocessed as described above and analyzed by ultra-performance liquidchromatography-electrospray ionization-tandem mass spectrometry(UPLC-ESI-MS) based on the procedures described in the art (see Qu etal., 2013, Immunobiology 218:496-505; Primikyri et al., 2017, JChromatogr B Analyt Technol Biomed Life Sci 1041-1042:19-26). The methodwas slightly adjusted for the analysis of plasma samples containingmPEG(3k)-Cp40. Here, a fixed collision energy of 35 V was applied in theion trap. For the preparation of standard curves, the areas under thecurve (AUCs) of the respective MS peaks (triple-charged Cp40 andisotope-labeled Cp40, quadruple-charged Cp40-K (fragment), Cp40-KK andCp40-KKK, and the mono-charged fragment at 436.256 m/z of mPEG(3k)-Cp40(-Sar-Ala-His-Arg-)) were determined by integration and plotted againstthe concentration. The plasma concentration at each time point wascalculated from the extracted peak area of the same mass peaks of eachpeptide using the corresponding standard curve.

To determine the plasma half-life and additional pharmacokineticparameters, the half-life of the peptides in NHP plasma was determinedas described in Qu et al. (supra) and Primikyri et al. (supra). Themaximum concentration (c_(max)) and time of maximum concentrationobserved (t_(max)) were determined manually from the pharmacokineticprofiles. The AUC from 0-120 h (AUC_(0-t)), AUC from 0-infinite time(AUC_(0-∞)), the apparent volume of distribution Vz/F,(F=bioavailability), and the apparent clearance CL/F were calculatedusing the following equations:

${{AUC}_{0 - t_{n}} = {\int_{0}^{t_{n}}{c\mspace{14mu} t*{dt}}}},{{AUC}_{0 - \infty} = {{AUC}_{0 - t} + {AUC}_{t - \infty}}},{\frac{Vz}{F} = \frac{CL}{k_{el}}},{\frac{CL}{F} = \frac{DOSE}{{AUC}_{0 - \infty}}},$

with t_(n)=120 h, k_(el)=the elimination rate constant. Individualpharmacokinetic parameters were calculated using a non-compartmentalapproach with Phoenix 64 WinNonlin Build 8.0.0.3176 software. The plasmamodel (extravascular dosing) was used with linear trapezoidal linearinterpolation.

Immunonephelometry was carried out to determine the levels of complementcomponent C3 in NHP plasma, using the N antisera to Human ComplementFactors (C3c) assay kit (Dade Behring, Marburg, Germany). In order tovalidate the assay for C3 quantification in NHP plasma, C3concentrations were first measured in human and then in NHP plasma todetermine the differing degrees of reactivity of the antibodies in thekit. For this purpose, serial dilutions of NHP plasma were spiked withknown concentrations of purified C3 from cynomolgus monkeys. The C3concentrations determined in human and NHP plasma were correlated, and acorrection factor (CF) of 1.2 was obtained for the C3 concentration inNHP plasma. The C3 concentrations in NHP plasma c_(C3)* were measured bynephelometry and corrected to the final concentrations using theequation c_(C3)=c_(C3)·CF. C3 baseline levels and C3 levels during thecourse of the experiments were assessed using the described assay.

For the assessment of proteolysis of Cp40-KK and Cp40-KKK in NHP andhuman plasma and in NHP total protein skin tissue, Cp40-KK or Cp40-KKKwas spiked into either NHP or human plasma at a final concentration of16 μM. Cp40-KKK was also spiked into NHP skin total protein (fromcynomolgus monkeys; Zyagen, San Diego, Calif.) to a final concentrationof 20 μM. The samples were incubated for 24 h at 37° C. in a water bathand samples were taken before and at various time points during and atthe end of the incubation. The samples were subjected to proteinprecipitation as described above and analyzed by UPLC-ESI MS asdescribed above.

Results.

Each peptide was injected subcutaneously (sc) in a single dose (8 mg; 2mg/kg) into two monkeys, and blood samples were collected over a periodof 5 days (t=pre-injection, 5, 30 min, 1, 2, 4, 6, 12, 24, 48, 72, 96,120 h after injection, FIG. 6A). As shown in FIG. 3B and Table 4, thehalf-life of Cp40 was 41 h and 48 h for the two monkeys. In comparison,the pharmacokinetic profiles of mPEG(3k)-Cp40 administered as asingle-dose sc injection to two cynomolgus monkeys showed that a highermaximum concentration of mPEG(3k)-Cp40 was reached, and the half-lifewas extended (t_(1/2) 65 h and 53 h, respectively) (FIG. 6C and Table4).

TABLE 4 Pharmacokinetic parameters calculated from the pharmacokineticprofiles of Cp40, Cp40-KK, Cp40-KKK, and mPEG(3k)-Cp40 in NHP plasmaafter s.c. administration. CL/F c_(injection) t_(1/2) t_(max) C_(max)AUC_(0-t) AUC_(inf) Vz/F ((mg/kg)/ Analog Animal (mg/ml) (h) (h) (μM)(μM · h) (μM · h) (ml/kg) μM · h) Cp40 1 4* 40.9 2 6.20 233 263 4489467605 2 48.1 2 5.53 218 255 545665 7856 Cp40-KK (SUM) 1 4* 145 1 12.90329 597 700664 3348 2 276 1 9.26 257 755 1055443 2648 Cp40-KKK (SUM) 132** 46.7 6 11.90 658 805 167382 2483 2 41.8 2 15.10 573 685 176010 2921mPEG(3k)-Cp40 1 16* 64.8 6 11.10 654 922 202917 2169 2 53.7 2 8.99 529656 236127 3050 Abbreviations: t_(1/2), half-life; t_(max), time ofmaximum concentration observed; C_(max), maximum concentration; AUC,area under the curve; Vz/F, apparent volume of distribution (F =bioavailability); and CL/F, apparent clearance. *dissolved in saline**dissolved in 100 mM phosphate buffer.

In addition, when mPEG(3k)-Cp40 was administered the level of C3 wassaturated for a longer period of time (33-35 h, vs. 4-5 h for Cp40). Asingle s.c. injection of mPEG(3k)-Cp40 in cynomolgus monkeys resulted ina pharmacokinetic profile comparable to that obtained for administrationof the parental compound Cp40, with a t_(max) at 2 to 6 h post-injection(see FIG. 6B, C). Despite the similar pharmacokinetic curve,mPEG(3k)-Cp40 reached a C_(max) of about 10 μM, almost 2-fold higherthan that of the parental peptide (FIG. 6C and Table 4). Furthermore,the AUC0-120 h (˜592 μM h) was higher, the t_(1/2) (˜59 h) was longer,and the CL/F (˜2610 mL h-1 kg-1) was slower than the respective valuesobtained for Cp40 (see Table 4). In addition, the target protein C3 wassaturated for a longer period of time (˜34 h vs ˜4.5 h for Cp40) (FIG.6B, C, dotted lines).

FIG. 6D shows the pharmacokinetic profiles of Cp40-KK over time in theplasma of two cynomolgus monkeys injected sc with the compound. Cp40-KKreached its maximum concentration in the plasma of both animals faster(t_(max)=1 h) than did Cp40 (t_(max)=2 h), and a higher concentrationwas achieved; however, the plasma concentration of Cp40-KK alsodecreased faster than did that of Cp40 (FIGS. 6A and D and Table 4).Whereas Cp40 remained above the plasma level of C3 for 4-5 h, the plasmaconcentration of Cp40-KK was reduced to a level lower than that of C3after 2.5-5 h. In general, the peptide concentration seemed to depend onthe C3 concentration. The overall clearance of Cp40-KK was very slow,with a half-life of 145 h in one monkey and 276 h in the other. Inaddition to Cp40-KK, Cp40-K was also detected in the NHP plasma samples,beginning at 30 min after sc injection of Cp40-KK, indicating enzymaticcleavage of the terminal Lys residue of the peptide. However, the levelof Cp40-K remained almost unchanged over time (FIGS. 7A and B). SinceCp40-K resulted from Lys cleavage of Cp40-KK, the sum of theconcentrations of both conjugates are shown in FIG. 6.

Cp40-KKK was administered to two cynomolgus monkeys via a single-dose scinjection. Instead of saline, phosphate buffer was used to lower the pHof the Cp40-KKK solution to bring the pH into physiological range. Thesingle s.c. injection of Cp40-KKK resulted in a pharmacokinetic profilecomparable to that of Cp40 and mPEG(3k)-Cp40. For instance, the Cp40-KKKpeptide reached a C_(max) of about 13.5 μM at 2 to 6 h post-injection(FIG. 6E and Table 4), with an AUC0-120 h of ˜616 μM h, CL/F of ˜2700 mLh-1 kg-1, and t_(1/2) of ˜44.3 h. In addition, after the administrationof Cp40-KKK, plasma levels of C3 remained saturated for about 40 h,which was about 7-fold longer than the saturation period observed withunmodified Cp40 (FIG. 6E). These data indicate that the Cp40 derivativesmPEG(3k)-Cp40 and Cp40-KKK have an improved pharmacokinetic profileafter a s.c. injection when compared with the parental Cp40 compound.

Quantification in NHP plasma proved to be more tedious for Cp40-KKK thanfor the other peptide analogs. UPLC-ESI-MS analysis revealed asuspiciously low Cp40-KKK concentration in plasma, with values notexceeding 2 μM at any time point in both animals (FIGS. 7C and D).Superimposition of the BPI chromatograms of the NHP plasma samplesshowed increasing peaks at the retention times for Cp40-KK (t_(R)=4.35min) and Cp40-K (t_(R)=4.65 min) over time after injection of Cp40-KKK;the peak of the compound itself (t_(R)=4.10 min) was small from thebeginning Examples of these chromatograms are shown in FIG. 8. Theintensity of the internal standard, isotopically labeled Cp40, whichappeared at the same retention time as Cp40 (t_(R)=4.92 min), remainedunchanged, indicating that one or two Lys residues are cleaved fromCp40-KKK in NHP plasma, but not the third. Therefore, the concentrationsof Cp40-KKK, Cp40-KK, Cp40-K, and Cp40 was determined in all the plasmasamples from both animals.

As shown in FIGS. 7C and D, no Cp40 was detected in any of the plasmasamples. The lowest concentration was found for Cp40-KKK, indicatingthat most of the compound is immediately cleaved in plasma to formCp40-KK, although Cp40-KKK could still be detected over the entireperiod of study. In contrast to the plasma samples from monkeysreceiving Cp40-KK, a larger amount of Cp40-KK was further cleaved toCp40-K; in one monkey, as much as 5 μM Cp40-K was detectable 4 h afterthe injection of Cp40-KKK (FIG. 7D). The amount of Cp40-K, Cp40-KK, andCp40-KKK was summed to assess the overall concentration of peptide inNHP plasma at each time point (FIG. 6E and FIG. 7). The half-life ofCp40-KKK (42 h and 47 h for the two monkeys) was comparable to that ofCp40 (Table 4). Nevertheless, the maximum concentration of Cp40-KKK washigher and was reached at a later time than for Cp40, at least in oneanimal. In addition, the level of Cp40-KKK remained above the C3concentration six to nine times longer as did that of Cp40.

The cleavage of Cp40-KKK could also be observed after multiple s.c.injections in cynomolgus monkeys (FIG. 9). Most interestingly,accumulation of the levels of Lysine-containing derivatives was observedover time (FIG. 9). Such an “accumulation” phenomenon is intrinsic ofthe Cp40-KKK analog, as it was not previously observed after multiples.c. injections of the parental Cp40 compound (FIG. 10). Of note, theobserved C_(max) of Cp40 after multiple s.c. injections did not exceed8-10 μM even when the compound was dosed in cynomolgus monkeys asfrequently as every 8-12 hrs (FIG. 10). Conversely, the observed C_(max)of an identical dose of Cp40-KKK, after multiple s.c. dosing, wasmarkedly higher, reaching or even exceeding 20 μM. In addition, theplasma bioavailability of the Cp40-KKK analog was markedly higher eventhough the compound was dosed through the s.c. route at less frequentintervals than Cp40 (i.e., every 48 hrs), which indicates that theunique structure of Cp40-KKK may confer new and beneficialpharmacokinetic properties to this compound that contribute to itsprolonged residence, increased bioavailability, and sustained inhibitoryactivity.

Example 5. Pharmacokinetic Properties of Cp40-Based Analogs afterIntravenous Administration

In vivo pharmacokinetic studies were conducted, in which cynomolgusmonkeys were administered single i.v. injections of mPEG(1k)-Cp40,mPEG(3k)-Cp40, Cp40-KK, or Cp40-KKK. The pharmacokinetic profiles of theCp40 analogs are summarized in Table 5 and FIG. 11.

TABLE 5 Pharmacokinetic profiles of Cp40 analogs following i.v.injection. PK Cp40-analogs parameters PEG(1k)-Cp40 PEG(3k)-Cp40 Cp40-KKCp40-KKK t_(1/2) (h) 53.2 84.37 49.23 35.31 AUC 0-120 304.4 313.59 85.39312 (μmol/L*h)

The i.v. injection studies revealed that mPEG(1k)-Cp40, mPEG(3k)-Cp40,and Cp40-KKK have a higher AUC0-120 h (304.4-312 μM h) when compared tothe Cp40-KK analog (85.23 μM h) (see Table 5 and FIG. 11).Interestingly, the Cp40-KKK analog displayed the shortest t_(1/2) of theCp40 analogs tested (see Table 5).

At the same time, while mPEG(1k)-Cp40, Cp40-KK had similar t_(1/2)values, mPEG(3k)-Cp40, in comparison, had an extended t_(1/2) by atleast ˜30 h (Table 5). The extended t_(1/2) of mPEG(3k)-Cp40 points tothe potential for the advancement of this analog as a therapeutic fori.v. treatment of systemic complement-mediated conditions.

Interestingly, as shown in example 4, UPLC/ESI-MS-based quantificationof peptide cleavage fragments of Cp40-KK or Cp40-KKK in the plasma ofs.c.-injected cynomolgus monkeys revealed that Cp40-KK is cleaved invivo generating minimal amounts of Cp40-K, while the Cp40-KKK analog isalmost completely metabolized into Cp40-KK and small amounts of Cp40-K(see FIG. 7). These data suggest a rapid cleavage of Cp40-KKK to Cp40-KKupon s.c. injection and indicate that the largest proportion of activecompound in NHP plasma is represented by the Cp40-KK metabolic species).

Similarly, upon i.v. injection, Cp40-KK was also metabolized to minimalamounts of Cp40-K (see FIG. 12). Further, at 12 h post-injection, mostof the Cp40-KKK analog was also metabolized into Cp40-KK and smallamounts of Cp40-K (see FIG. 12). Accordingly, the data show that Cp40-KKis the main compound remaining in the circulation upon injection ofeither Cp40-KK or Cp40-KKK. FIG. 18 is a summary of the in vivo and invitro Lys-cleavage in Cp40-KK and Cp40-KKK.

Example 6. Pharmacokinetic Properties of Cp40-Based Analogs afterIntramuscular Administration

The pharmacokinetic profiles of Cp40, Cp40-KK and Cp40-KKK were testedin vivo after a single i.m. injection of 100 mg of compound, equivalentto 25 mg/kg (FIG. 13). Notably, the compounds show a differentpharmacokinetic profile after i.m injection in relation to the profileobserved after i.v. or s.c. injections, thus suggesting that the i.m.administration route may be exploited in novel dosing protocols for thetailored delivery of these compounds in certain complement-mediatedindications. Most interestingly, the t_(1/2) after a single i.m.injection of these analogs is about 8 days (FIG. 13). There observationssuggest that after an initial s.c. or i.v. injection of the disclosedCp40 analogs to saturate target levels of the compound, subsequent i.m.injections may be used as a maintenance dose (e.g., administered viai.m. every two weeks).

Example 7. Cp40 and Cp40 Lysine Analog Antibody Generation andSpecificity

To generate antibodies against compstatin analogs for the WES and SPRdetection methods described in Examples 8 and 9, rabbits (n=2/analogue)were immunized with 100 μg of Cp40 or other Lysine analogs conjugatedwith Keyhole limpet hemocyanin (KLH) in the presence of a strongadjuvant (TiterMax Gold-100 μl) followed by weekly injections of 50 μgof KLH-Cp40 plus adjuvant for a total of 5 weeks. Antibody productionwas monitored with a direct ELISA whereby Cp40-analogs (10 μg/ml) werecoated on a 96-well plate, followed by blocking with PBS/1% BSA andincubation with serial dilutions of pre- and post-immunization plasma.The assay was developed after incubation with anti-rabbit IgG conjugatedwith horseradish peroxidase (HRP), addition of appropriate chromogenicsubstrate and measurement of absorbance at 405 nm using an ELISA platereader. This immunization procedure resulted in the production of ananti-Cp40 and an anti-Lys analog antibody (AB101 and AB102,respectively) that were further purified from the rabbit serum byaffinity chromatography with Protein A. ELISA and Western blot data showthat these antibodies are highly specific as they only react withbiological samples (plasma, vitreous) containing the same peptides thatwere used as immunogens (see FIG. 14 for ELISA. The AB102 antibodyshowed the same specific for CP40-K, CP40-KK, CP40-KKK. The antigenicepitope of AB101 requires the C-terminal mIle of Cp40 and that of AB102the C-terminus Lysine residues. Thus these characteristics unique fordetecting the CP40 and its analogs.

Example 8. Residence Time of Cp40-Based Analogs in the Vitreous ofNon-Human Primates

To determine the residence time of mPEG(3k)-Cp40, mPEG(1k)-Cp40,Cp40-KK, and Cp40-KKK in the vitreous of cynomolgus macaques, theseCp40-based analogs were administered intravitreally (i.v.t.) to theanimals after induction of light ketamine anesthesia (10-15 mg/kg, byintramuscular injection (i.m.)) and dilation of the pupils withtropicamide/phenylephrine ophthalmic solution (see Table 6 for studydesign).

TABLE 6 Study design for intravitreal administration and detection ofCp40-based analogs. Dose N Group Animal No. Eye Treatment (μg/eye)(eyes) G1 K-784, K-792, Right Cp40-KK 500 3 G2 K-799 Left Cp40-KKK 500 3G3 K-780, K-786, Right PEG(1K)- 500 3 K-800 Cp40 G4 Left PEG(3K)- 500 3Cp40 G1, G2, G3, G4: Time point of sample collection and photography 0Pre- 0 56 (G1, 73 (G1, 90 (G1, Day dose Dosing 14 28 42 G2) G2) G2)Vitreous — — x x x x x x fluid Photography x x x x x x x x

For i.v.t. injection, the cynomolgus monkeys were anesthetized with acombination of ketamine (15-25 mg/kg, i.m.) and xylazine (2 mg/kg, i.m.)and the eye was cleaned with povidone-iodine solution. After applicationof oxybuprocaine hydrochloride solution (Benoxil® ophthalmic solution0.4%) to the cornea as a local anesthetic, the Cp40-based analogs wereadministered to the animals by i.v.t. injection into either the right(G1, G3) or left (G2, G4) eye of each animal. Immediately following eachinjection, a single topical dose of 0.5% levofloxacin was administered.After i.v.t. injection of mPEG(3k)-Cp40, mPEG(1k)-Cp40, Cp40-KK, andCp40-KKK, vitreous fluid samples (about 50 μL each) were withdrawn fromCp40-based analog-treated eyes on days 14, 28, 42, (G1, G2, G3, G4), 56,73, 90 (G1, G2) (this was done either three times or six times). Sampleswere collected on ice and stored in a deep freezer (−79.3 to −68.5° C.)until further analysis. On day 73 and 90, about 100 μL of vitreous fluidfrom G1 and G2 were withdrawn. Presence and concentration of theCp40-based analogs was determined using either the simple westerntechnology (WES, ProteinSimple, San Jose, Calif.), or surface plasmonresonance (SPR)-based real-time measurement of binding kinetics on aCp40-KKK immobilized chip (see detailed methods below). Similar resultswith regard to the intravitreal residence time and concentration levelsof the test compounds were obtained regardless of the quantificationmethod selected.

To quantify the levels of all test compounds in vitreous samplescollected at different time points following i.v.t. injection (see FIG.15), WES analysis was performed on a WES instrument (ProteinSimple, SanJose, Calif.) using a 2 to 40 kDa Separation Module and Anti-RabbitDetection Module according to the manufacturers' instructions. In brief,undiluted eye vitreous samples were mixed with a Fluorescent Master Mixand heated at 95° C. for 5 min. The samples, blocking reagent, primaryantibodies (in house developed anti-Cp40; 1:1000 in blocking reagent),HRP-conjugated secondary antibody (anti-rabbit IgG) and chemiluminescentsubstrate were pipetted into the plate (part of Separation Module). Theused instrument settings were: stacking and separation at 375 V for 27min; blocking reagent for 30 min, primary and secondary antibody bothfor 30 min; Luminol/peroxide chemiluminescence detection for ˜15 min(exposures of 5, 15, 30, 60, 120, 240, and 480 s). The resultingelectropherograms were evaluated using the Compass software(ProteinSimple, San Jose, Calif.). The following criteria were used todiscriminate low peptide signals from background: The peaksignal-to-noise (S/N) ratio given by the software must be ≥10, and thepeak height/baseline ratio (calculated manually from the peak height andbaseline values given by the software) must be equal to or greater than3. A five-point standard curve of known concentrations (100-2,000 nM) ofpeptide spiked in rabbit vitreous was used to calculate peptideconcentration in the vitreous samples using the Compass software(Compass Software Inc., Atlanta, Ga.). The curve showed an R² greaterthan 0.98. Shown in FIG. 15 is a representative standard curve generatedusing the WES system and bands obtained upon running pre-determinedamounts of purified peptide spiked into rabbit vitreous samples.

To corroborate the WES-based measurements for all test compounds, anSPR-based competition assay was developed for detecting Cp40 and Cp40analogs in biological samples. Briefly, plasma samples diluted 1/1000were used for testing with or without the addition of Cp40 or Cp40analogs. The samples were heated at 95° C. for 5 min. After heattreatment, the samples were spun and the supernatant was mixed with afixed concentration of C3 or plasma as a source of C3 and flown over aCM5 sensor chip to which the corresponding Cp40 analog was immobilized(e.g., covalently attached). The response was then measured and comparedto a standard curve for quantitation. A schematic of the SPR-basedmethod is summarized in FIG. 16.

The novel SPR-based competition assay was performed on the same vitreoussamples using a Biacore 3000 instrument (GE Healthcare, PiscatawayTownship, N.J.) at 25° C. Cp40-KKK was covalently attached to a CM5sensor chip using standard amine coupling reactions with HBS-EP asrunning buffer. Briefly, the chip was activated for 7 min with NHS/EDC(1:1), coated with Cp40-KKK using 300 μg/ml Cp40-KKK in 5 mM sodiumacetate (pH 5.0) for 6 min, and deactivated with 1 M ethanolamine-HCl(pH 9.5) for 10 min. An empty flow cell served as reference surface. Inorder to eliminate non-specific binding during the experiments, therunning buffer was changed to 50 mM phosphate buffer (pH 7.4) containing100 mM NaCl, 0.05% Tween 20, 10 mM EDTA and 1 mg/ml dextran sulfate (500kDa). Experiments were all carried out at a flow rate of 10 μl/min with2 min of sample injection followed by regeneration of the surface of thesensor chip. The surface was regenerated with subsequent injection of0.5% SDS for 1 min and 50 mM glycine buffer (pH 9.5) for 30 s.

Depicted in FIG. 17 is a representative standard curve of the relevantCp40-based analog that was prepared by diluting pre-determinedconcentrations of peptide with rabbit vitreous followed byheat-inactivation for 5 min at 95° C. and 10 min equilibration in a roomtemperature water bath. The standard samples were centrifuged for 10 minat 14,000×g and supernatants were mixed with normal human plasma.Similarly, for the detection of unknown concentrations of mPEG(3k)-Cp40,mPEG(1k)-Cp40, Cp40-KK, and Cp40-KKK in eye vitreous, samples werediluted in running buffer followed by heat-inactivation, equilibration,and centrifugation as described above. Sample supernatants were mixedwith diluted human plasma as described above and injected. Dataprocessing was performed using the Scrubber software (BioLogic Software,Campbell, Australia).

As shown in FIG. 18, Cp40-KK, Cp40-KKK, PEG(1K)-Cp40, PEG(3K)-Cp40 weredetected in the vitreous collected from treated animals 14 days after asingle 0.5 mg injection of the Cp40-based analog. It should be notedthat the PEG(1K)-Cp40 and PEG(3K)-Cp40 compounds were detected at day 14post injection but not in later time points (see FIG. 18), indicatingthat the vitreous residence time of PEGylated-compounds is at least 14days, but significantly shorter than that of the lysine-containingcompounds (i.e., Cp40-KK and Cp40-KKK).

The Cp40-KK and Cp40-KKK peptides were detected at days 14, 28, 42, 56,73 and 90 post-injection (FIG. 18). While the concentration of CP40-KKin the NHP vitreous decreases over time, from days 42 to 73,concentration levels of Cp40-KKK remained stable during the 73-dayobservation period (FIG. 18). Of note, Cp40-KKK showed the longestresidence time in the ocular tissues as it was detected even at day 90post-treatment. Conversely, the residence time of Cp40-KK in thevitreous was markedly shorter than that of Cp40-KKK, as it was no longerdetected on day 90. It should be noted that the intravitrealconcentration of Cp40-KKK reached and maintained saturating levels withregard to those of its target C3 in the intravitreal compartment (0-140nM; see Loyet K M et al., 2012, Invest Ophthalmol Vis Sci. 53:6628-37)during the entire observation period leading up to day 90 (see FIG. 18,dotted line).

While the addition of mini-PEG moieties or the extension of a peptidewith charged, hydrophilic residues, such as Lysine, are both chemicalmodifications known in the art for their capacity to increase apeptide's solubility, the markedly increased residence time of Cp40-KKand Cp40-KKK in ocular tissues was a very surprising result and had notpreviously been reported. The prolonged ocular residence of the testcompound (Cp40-KK and Cp40-KKK) is unique to its structure and, whilenot intending to be bound by theory, may be attributed to atissue-specific mechanism exploiting the presence of the tandem Lysrepeat at the C-terminus of the compound. This is further supported bythe observation that while the mini-PEGylated Cp40 exhibits comparablesolubility profiles with the Cp40-Lys (n) compounds (see Table 1), ithas distinctly shorter ocular residence than Cp40-KK or Cp40-KKK.Moreover, the unexpected and significant increase in ocular residenceafforded by the addition of a third Lys residue to Cp40-KKK as comparedto the Cp40-KK derivative cannot merely be explained by taking intoaccount their similar solubility profiles (see Table 1), but ratherpoints to a novel pathway by which the Cp40-KKK is retained in thevitreous humor for a longer period even as compared to the Cp40-KKanalog.

Of note, the amount of compounds detected in the vitreous of treatedcynomolgus monkeys on day 14 represents approximately 0.2% of theinitially injected dose, which suggests a biphasic, target-drivenelimination profile whereby the compound in excess of the target C3concentration is rapidly cleared from the eye and only the compound thatis tightly bound to its target remains longer in the tissue. In otherwords, i.v.t. administration with these Cp40-based analogs can achievethe observed residence time at much lower doses (e.g., even less thanabout 10 g or even 1-2 g). Conversely, when 100 g of Cp40 was i.v.tadministered no detection of any compound was observed after one monthsuggesting that the residence time of Cp40 is very short as compared tothe Cp40-based analogs discussed herein. Therefore, the disclosedCp40-based analogs confer a beneficial increase in residence time inaddition to enhanced solubility as compared to Cp40.

In summary, the data above show that the intravitreal residence time ofCp40-KK is less than Cp40-KKK, and the residence time of Cp40-KKK isequal to or exceeds about 3 months after i.v.t. These findings haveimportant implications for the design of chronic administrationprotocols for the treatment of ocular diseases and other clinicalpathologies with involvement of deregulated complement activation. Theprolonged ocular residence of Cp40-KK and Cp40-KKK will enable theapplication of new drug dosing schemes that involve a significantlyreduced dosing frequency with lesser patient burden, compared to that ofthe previous generation compounds analogs Cp40 and APL-2.

Example 9. Activity of Lysine-Containing Cp40-Based Analogs in theVitreous of Non-Human Primates

To confirm that the compound present in the NHP vitreous maintainsC3b-binding activity, which is a prerequisite for eliciting itsinhibitory action across all complement pathways, surface plasmonresonance (SPR) binding experiments were conducted. Briefly, for apositive binding control, serial dilutions of Cp40-KKK (16, 8, 4, and 2nM) were added to rabbit vitreous samples and flowed onto a Biacore CM5chip immobilized with purified human C3b (Complement Technology Inc,Tyler, Tex.) (see FIG. 19A). Next, vitreous samples were collected froma Cp40-KKK injected eye at day 14 post-injection and subjected toheat-inactivation at 95° C. for 5 min. Both heat-inactivated andnon-heat-inactivated samples were flowed onto a CM5 chip immobilizedwith purified human C3b as indicated above (FIG. 19B).

As shown in FIG. 14A, Cp40-KKK spiked into rabbit vitreous samples bindsto C3b in a dose-dependent manner. Further, signal for C3b binding isobserved upon the testing of vitreous samples collected from an eyeinjected with Cp40-KKK (FIG. 19B), indicating that Cp40-KKK present inthe vitreous maintains its C3b-binding activity.

Notably, the C3b-binding signal obtained with a vitreous sample that washeat-inactivated (FIG. 19B, left bar) was higher when compared with thesignal resulting from the non-heat inactivated (FIG. 19B, right bar)sample (10 vs. 2 relative units, respectively). As the heat-inactivationstep assures the dissociation of the compound from its target, thedifferential response observed in the SPR analysis likely indicates thatthe majority of the compound is bound to the complement C3 protein inthe vitreous. This observation is in concordance with the high-affinity,tight binding of Cp40 and its derivatives (Cp40-KKK, Cp40-KK) to C3 andaligns with the biphasic target-driven elimination profile of thesecompounds, as previously reported in NHP studies (see Risitano et al.,2014, Blood 123(13):2091-2101; Berger et al., 2018, J Med Chem61(14):6153-6162).

An alternative explanation to the one presented above is that thevitreous contains additional unidentified factors that associate withthe Cp40-KKK analog. To investigate whether an unidentified vitreousfactor impacts the complement inhibitory activity of lysine containingcompounds, complement activation assays were performed using vitreoussamples. Briefly, human plasma was incubated with OVA-anti-OVAimmune-complexes in the presence of serial dilutions of the Cp40-basedanalogs mixed with rabbit vitreous. Levels of classical pathwayactivation (C3b deposition) were determined by ELISA using a polyclonalanti-human C3 antibody conjugated with HRP (MP Biomedicals, Solon, Ohio)(FIG. 20).

As shown in FIG. 20, in the absence of vitreous, all the compoundstested (Cp40, Cp40-1K, Cp40-KK, Cp40-KKK) show complement classicalpathway-mediated inhibitory activity in a dose-dependent manner (solidlines). Notably, the vitreous samples appear to have a minor complementinhibitory activity as the presence of vitreous appears to potentiatethe complement inhibitory activity of the tested compounds (FIG. 20,compare dashed lines with solid lines). Thus, the data indicate that theactivity of tested compounds (i.e., Cp40 and its lysine-containingderivatives) is not inhibited by unidentified factors in the vitreousthat may bind to some extent these compounds

Summary: Comparison of the PK Properties of Individual Analogues ViaDifferent Administration Routes and their Potential for the Developmentof Therapeutics.

The data shown in Example 8 indicated that the Cp40-KKK and Cp40-KKcompounds exhibit improved residence time in the eye vitreous whencompared with other tested compounds while maintaining their bindingactivity, hence supporting the potential for development of thesemolecules as therapeutics for ocular conditions. Additionally,pharmacokinetic data obtained from subcutaneous (s.c.) and intravenous(i.v.) studies in cynomolgus monkeys indicated that mPEG(1k)-Cp40,mPEG(3k)-Cp40, Cp40-KK, and Cp40-KKK have discrete pharmacokineticproperties from their parental molecule, Cp40, and, by virtue of thesepharmacokinetic properties, have unique potential to be developed asboth locally and systemically administered therapeutics for modulatingcomplement activity in clinical disorders (see FIGS. 6 and 11; Table 4).

Specifically, s.c. administration of the Cp40-KKK compound saturates C3plasma levels for about 40 h, which is about 7-fold longer than thesaturation period observed with unmodified Cp40 (Example 4; FIG. 6E).The long target saturation period of Cp40-KKK, in combination with itsincreased solubility, confers a unique potential for development as as.c. therapeutic, suitable for the treatment of systemic and/or chroniccomplement-mediated conditions. The unique structure of the Cp40-KKKanalog may dictate its prolonged half-life and likely enhancedbiodistribution in different compartments, such as the eye vitreous, dueto potential depot effects that may promote its accumulation during amultiple dosing regimen and its slower release into the targeted tissuethrough potential interactions with other unknown carrier-like orbinding proteins. Moreover, the observation that a significant amount ofthe Cp40-K metabolite was detected in the plasma of Cp40-KKK-injectedcynomolgus monkeys (FIG. 12), whereas this metabolite was almost absentfrom the plasma of CP40-KK-injected animals, indicates that the Cp40-KKmetabolite may likely be more stable in the circulation (e.g., lessprone to further proteolytic degradation) and that the addition of athird lysine residue to the C-terminus of the parental Cp40 confers anew biotransformation profile and novel pharmacokinetic properties tothe resulting peptide that collectively enhance its plasma andintravitreal residence. These characteristics thus afford the Cp40-KKKanalog a more favorable pharmacokinetic profile that can enable chronicadministration of this analog with less frequent dosing intervals. Shownin FIG. 21 is a summary of the in vivo and in vitro Lys-cleavage inCp40-KK and Cp40-KKK.

Similarly the extended t_(1/2) of mPEG(3k)-Cp40, observed after i.v.injection in NHP (Example 5; Table 5), points to the potential for theadvancement of this analog as a therapeutic for i.v. treatment ofsystemic complement-mediated conditions. Moreover, as shown in example6, the i.m. administration route may be exploited in novel dosingprotocols for the tailored delivery of the Cp40 analogues incomplement-mediated indications.

The present invention is not limited to the embodiments described andexemplified herein, but is capable of variation and modification withinthe scope of the appended claims.

What is claimed:
 1. A compound comprising a peptide having an amino acidsequence of SEQ ID NO: 9 or SEQ ID NO:
 10. 2. The compound of claim 1,further comprising a polymer having an average molecular weight of about3 kDa or less.
 3. The compound of claim 2, wherein the polymer ispolyethylene glycol (PEG).
 4. The compound of claim 3, wherein the PEGis linked to the N- or C-terminus of the peptide.
 5. The compound ofclaim 3, wherein the PEG is a monodisperse PEG having a molecular weightof about 0.5 kDa to about 3 kDa.
 6. The compound of claim 3, wherein thePEG is a polydisperse PEG having an average molecular weight of about0.5 kDa to about 3 kDa.
 7. A pharmaceutical composition comprising thecompound of claim 1 and a pharmaceutically acceptable carrier.
 8. Thepharmaceutical composition of claim 7, formulated for administration toan individual by a route selected from the group consisting of: oral,intranasal, topical, intraocular, periodontal, pulmonary, subcutaneous,intramuscular and intravenous.
 9. The pharmaceutical composition ofclaim 8, wherein: (a) the intraocular administration is intravitrealadministration; or (b) the periodontal administration is gingivaladministration or intrapapillary infiltration injection of the compound.10. A peptide consisting of SEQ ID NO:9 or SEQ ID NO:10.
 11. A method oftreating an individual having a pathological condition associated withcomplement activation, the method comprising the steps of: (1) providingthe individual having the pathological condition associated withcomplement activation; (2) administering to the individual atherapeutically effective amount of a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a compoundcomprising a peptide having an amino acid sequence of SEQ ID NO: 9 orSEQ ID NO: 10; and (3) measuring one or more parameters of complementactivation or of the pathological condition in the individual; whereinadministering of the pharmaceutical composition results in inhibition ofcomplement thereby treating the individual having the pathologicalcondition associated with complement activation.
 12. The method of claim11, wherein the pathological condition associated with complementactivation is selected from the group consisting of: atypical hemolyticuremic syndrome (aHUS); dense deposit disease (DDD); C3glomcruloncphritis (C3GN); C3 glomerulopathies; complement-mediatednephropathies and glomerular inflammatory diseases; age-related maculardegeneration (AMD); eye disorder characterized by macular degeneration,choroidal neovascularization (CNV), retinal Neovascularization (RNV),proliferative vitreoretinopathy, glaucoma, uveitis, ocular inflammation,or any combination of these; paroxysmal nocturnal hemoglobinuria (PNH);cold agglutinin disease (CAD); warm antibody autoimmune hemolyticanemias (wAIHAs); sickle cell disease; transplant-associated thromboticmicroangiopathies; rheumatoid arthritis (RA); systemic lupuserythematosus (SLE); autoimmune and autoinflammatory kidney diseases;autoimmune myocarditis; multiple sclerosis; traumatic brain and spinalcord injury; cerebral, intestinal and renal ischemia-reperfusion (IR)injury; spontaneous and recurrent pregnancy loss; anti-phospholipidsyndrome (APS); Parkinson's disease; Alzheimer's disease;neurodegenerative inflammatory conditions underpinned by aberrantsynaptic remodeling, microglial activity and cognitive decline; asthma;anti-nuclear cytoplasmic antigen-associated pauci-immune vasculitis(Wegener's syndrome); non-lupus autoimmune skin diseases such aspemphigus, bullous pemphigoid, and epidermolysis bullosa; post-traumaticshock; cancer; periodontitis; gingivitis; and atherosclerosis.
 13. Themethod of claim 11, wherein the pharmaceutical composition isadministered intravenously or subcutaneously at a therapeuticallyeffective dose of between about 0.125 mg/kg and about 10 mg kg.
 14. Themethod of claim 11, wherein the pharmaceutical composition isadministered intramuscularly at a therapeutically effective dose ofbetween about 0.25 mg/kg and about 50 mg/kg.
 15. The method of claim 11,wherein the pharmaceutical composition is administered intravitreally ata therapeutically effective dose of between about 1 μg and about 10 mg.16. The method of claim 11, wherein the pharmaceutical composition isadministered periodontally at a therapeutically effective dose ofbetween about 1 μg and about 1,000 μg.
 17. The method of claim 11,wherein the pharmaceutical composition is administered orally at atherapeutically effective dose of between about 1 mg and about 20 mg.18. The method of claim 11, wherein the pharmaceutical composition isadministered in regular intervals ranging from once every 12 hours toonce every 3 months.
 19. The method of claim 11, wherein thepharmaceutical composition is administered intravenously orsubcutaneously to the individual at a first therapeutically effectivedose of between about 0.125 mg/kg and about 10 mg/kg, and wherein thepharmaceutical composition is further administered: (i) intramuscularlyat a second therapeutically effective maintenance dose of between about0.25 mg/kg and about 50 mg/kg; or (ii) orally at a secondtherapeutically effective maintenance dose of between about 1 mg/kg andabout 20 mg/kg.
 20. The method of claim 11 wherein the individual is ahuman or a non-human primate.